Wear resistant antimicrobial compositions and methods of use

ABSTRACT

Anti-microbial compositions comprising (a) a quaternary ammonium compound and (b) a cationic biocide. Applicant has discovered a synergistic combination of the two components with the ration of cationic biocide to quaternary ammonium being less than 1:10 or with a single quaternary compound in a ratio of less than 1.6 to 1 provide a film forming coating that has residual anti-bacterial activity and improved durability with strong resistance to general wear between applications.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. §119 to applicationSer. No. 13/314,264 filed Dec. 8, 2011, which claims the benefit ofprovisional Application Ser. No. 61/422,908 filed Dec. 14, 2010, both ofwhich are herein incorporated by reference in their entireties.

FIELD OF THE INVENTION

This invention relates to anti-microbial compositions and use of thesame to disinfect or clean various surfaces.

BACKGROUND OF THE INVENTION

Microbes may often be present on many common objects and surfaces ineveryday life. Microbes can include, for example, bacteria, fungi,spores, viruses, prions, microorganisms such as, e.g., Mycobacteriumtuberculosis, listeria monocytogenas, escherichia coli, pseudomonasaeruginosa, salmonella typhimurium, salmonella enteritidis, legionellabacteria, Yersinia pestis, staphylococcus aereus, bacillus subtilis,enterobacter aerogenes, streptococcus faecalis, legionella pneumophila,vibrio parahaemolyticus, bacillus cereus, and other gram positive andgram negative microorganisms. Several such microbes/microorganisms,individually or in combination, can cause illness or other healthproblems, for example, when they come into contact with humans and/oranimals, or when they are ingested along with food which has contactedthem.

These microbes present health hazards due to infection or contamination.When microorganisms are present on the surface of a substrate they canreplicate rapidly to form colonies. The microbial colonies form acoating on the substrate surface, which is known as a biofilm. Biofilmsfrequently consist of a number of different species of microorganismswhich in turn can be more difficult to eradicate and thus more hazardousto health than individual microorganisms. Some microorganisms alsoproduce polysaccharide coatings, which makes them more difficult todestroy. The microorganisms attach themselves to substrates forming abiofilm comprising a “calyx” of polysaccharides and/or similar naturalpolymers as the affixing mechanism. Without this affixing point, thereproduction of the microorganism particularly bacteria cannot proceed,or is at least seriously impaired.

The health care and medical industry has an acute need formicro-organism-resistant surfaces. For example, hospitals and othermedical facilities may have a particular need for sterile anduncontaminated surfaces, both in surgical areas as well as inconvalescence facilities, where patient exposure may be significant andresistance to such microbes may be lowered. Much time and effort can bespent, for example, on sterilizing medical instruments, testing devices,etc. Often, such devices can be provided with disposable components orcovers (e.g., disposable thermometer probes) to avoidcross-contamination between patients. Disposable needles are alsocommonly used. Such disposable materials involve increased costs andincreased waste, as well as potential safety issues associated withtheir disposal.

The food-preparation and delivery industry is another area in whichpresence of microbes (e.g., bacteria) can be problematic. Foodpreparation facilities, if contaminated with microbes, can lead tocontamination of food which may cause health problems when ingested. Forexample, restaurants, food manufacturing plants, and even home kitchenscan contain preparation surfaces, utensils, and equipment which maycontaminate food that comes into contact with them. There may be, forexample, a particular need for reducing a presence and spreading ofmicrobes in meat packaging plants.

Public and private facilities such as, e.g., restrooms, may also containsurfaces which can harbor and spread microbes, leading to potentialhealth problems. To address this issue, products such as antimicrobialsoaps and air dryers for hands may be offered, as well as disposablepaper towels. Nevertheless, microbes may still be harbored on suchobjects as faucet and toilet handles, door knobs, keys, dispenserlevers, etc.

In the transportation industry, including land, sea, air, and spacevehicles, there may also be particular surfaces which can harbor andspread microbes, leading to potential health problems. For example,rental cars may benefit from durable antimicrobial surfaces (bothinterior and exterior). In particular, isolated environments such as,e.g., airplanes and submarines can also be safer if provided withantimicrobial surfaces.

Other common objects may benefit from antimicrobial compositions, whichcan inhibit or prevent spread of microorganisms between people and/oranimals that come into contact with such objects. For example, musicalinstruments, such as harmonicas, flutes, clarinets, etc., computerperipherals, communications equipment such as, e.g., telephones, petaccessories such as leashes and carriers, and/or other common householdobjects could benefit from antimicrobial surfaces.

Materials and procedures have been developed to reduce or prevent apresence of microbes on certain surfaces. For example, compounds whichmay exhibit antimicrobial activity such as, e.g., certain salts ornanoparticles of silver, can be applied onto or incorporated intocertain substrates. Such antimicrobials may be capable of killing orinhibiting growth of certain microbes. Application of such antimicrobialcoatings may often be performed using solution chemistry or by combiningantimicrobial compounds with polymers, resins, or other materials aspart of the casting or manufacturing process whereby those compounds maybe incorporated into the final product producing a “treated article”.Such materials may often be at least partially organic. However, suchcoatings may have limited lifetimes for killing or inhibiting growth ofmicrobes. More importantly, such coatings may often not provide a rapidand high level of microbial efficacy which could reduce the risk ofmicrobial contaminants being transferred from one surface to another.Further, such antimicrobial coatings may wear off to some degree whenexposed to various environments or conditions (e.g., heat, abrasion,chemicals). Such wear, which can occur rapidly, may reduce or eliminatethe ability of these coatings to kill or inhibit growth of microbes overtime.

One can see that there is a continuing need for improved antimicrobialcleaners, coatings, and sanitizers which are durable and effective inrapid and high level killing or inhibiting growth of microbes such asbacteria and other microorganisms.

There is a need to provide such materials and coatings which are easyand relatively inexpensive to produce, which have a long life time ofkilling, which do not wear off easily, and which may be applied to abroad variety of substrates.

In addition, there is a need for such antimicrobial coatings which canbe applied to objects that are already in use (clean in place) or thatare in need of repair

The present invention provides anti-microbial compositions which addressone or more of the aforementioned needs as well as others which willbecome apparent form the description of the invention which follows.

SUMMARY OF THE INVENTION

The anti-microbial compositions of the invention provide a film that islong lasting with residual antimicrobial effect with improved wearresistance when applied to a surface. The compositions comprise asynergistic blend of a quaternary ammonium compound, preferablyquaternary ammonium chloride, and cationic biocide such aspolyhexamethylene biguande. In preferred embodiments that compositionsalso include an amine oxide surfactant (preferably having a carbon chainlength of 8), and a chelant (Ethylenediaminetetraacetic acid EDTA) toproduce a bacterial/viricidal film that provides both wet efficacy (e.g.when the solution is applied directly to a surface) as well aspersistent antibacterial/antiviral activity that is significantly moreresistant to mechanical abrasion than either of the components alone orin various combinations.

According to the invention the applicants have discovered criticalranges and ratios of the various components that can form a synergisticinteraction and improve the anti-microbial coatings of the invention forenhanced wet efficacy, durability and residual anti-microbial activitywithout causing eye irritation under typical use rates. For example,Applicants have found that a composition of cationic biocide andquaternary ammonium with a ratio of the two components being less than1:10 (respectively), on an actives weight basis, provides a film formingcoating that has residual anti-bacterial activity and improveddurability with strong resistance to general wear between applications.In a more preferred embodiment the ratio is about 1:2.5 to about 1:10and most preferred is a ratio of about 1:2.5 to 1:6.5 of cationicbiocide to quaternary ammonium compound (See example #7)

Stated another way, in a use composition, applicants have discovered acritical range of about 1000 to about 6250 ppm quaternary ammoniumcompound and about 156 (Based on example #9 run @ 0.5 oz/gallon) to 2500ppm cationic biocide provides a film forming solution that retains itsantimicrobial activity and remains on the surface after numerousabrasion cycles. The compositions of the invention comprise at least onequaternary ammonium compounded with the polymeric biguanide.

Applicants surprisingly found that when additional functional componentswere added to the formulation, residual effectiveness against gramnegative organisms was negatively impacted and thus in order to retainenhanced wet efficacy and retain abrasion resistant residual activity,particularly against gram negative organisms, several criticalparameters were determined.

For example. Applicants found that as opposed to the larger classes ofsurfactants, such as alcohol ethoxylates, or sulfonated and sulfatedanionic surfactants, instead surfactants used in the compositions arepreferably amine oxides and more preferably amine oxides with a carbonchain length of 8. When formulations of the invention were prepared withother surfactants, such as alcohol ethoxylate, the efficacy of theformula was significantly reduced.

Thus Applicants have found that in order to keep efficacy, the ratio ofquaternary ammonium to surfactant should be from about 2:1 to about 7:1respectively on an actives weight basis.

Applicants have also found that the composition preferably includes achelant. Again here, Applicants have found that the chelant should be ina ratio of from about 2:1 to about 3.5 to 1 of quaternary ammonium tochelant respectively. In a preferred embodiment the chelant isethylenediaminetetraacetic acid.

In one embodiment the composition of the invention includes a quaternaryammonium compound, a cationic biocide, a chelant, and a surfactant ofamine oxide. In a preferred embodiment the invention includes aconcentrate composition including from about 1.25% to about 50% wt. % ofquaternary ammonium compound, from about 0.125% to about 8.0 wt. % ofcationic biocide, from about 0.4% to about 12.8 wt. % of amine oxide,and from about 0.4% to about 12.8% chelant.

In another aspect, the present invention relates to an anti-microbialformulation concentrate that, upon dilution with water, provides a readyto use formulation. In certain embodiments the concentrate compositionis diluted with water in a ratio of from about 1:10 to about 1:500,preferably from about 1:30 to about 1:400 and more preferably from about1:50 to about 1:260 parts of composition to diluent. In a preferredembodiment the above concentrate solution is diluted 1:64.

The invention includes a use solution comprising a use solution of fromabout 195 ppm to about 7800 ppm of quaternary ammonium compound, fromabout 19.5 ppm to about 1250 ppm of cationic biocide, from about 62.5 toabout 2000 ppm amine oxide, and from about 62.5 to about 2000 ppmchelant.

It is contemplated that formulations comprising the synergisticcombination disclosed above can contain additional ingredients asdescribed below and other ingredients that are standard in the art, thecompositions of the invention may consist of or consist essentially ofthe components listed in the paragraph above.

As will be appreciated, the percentage by weight of the components inthe compositions of the invention will depend to a large extent on theform in which a composition is provided and the intended use of acomposition. It is envisaged that the compositions will be made in aconcentrated form and then diluted to a suitable concentration for theintended use. More particularly, it is envisaged that commerciallyavailable solutions will include concentrated solutions which can bediluted by the user before use and ready diluted solutions that areready to use.

The important thing for compositions of the invention to provide therequired antimicrobial effect is not typically the concentration of thecomponents in the final solution, rather it is the ratio of the numberof molecules of the components. This ratio will remain the same whetherthe composition is in a concentrated form or whether it is in a dilute(ready-to-use) form.

By the term “anti-microbial” we mean that a compound or composition thatkills and/or inhibits the growth of microbes (microorganisms). The term“microbiocidal” is used to refer to compounds or compositions that killmicrobes. The compositions of the invention are anti-microbial and/ormicrobiocidal.

A microorganism or microbe is an organism that is microscopic (too smallto be seen by the human eye). Examples of microorganisms includebacteria, fungi, yeasts, moulds, mycobacteria, algae spores, archaea andprotists. Microorganisms are generally single-celled, or unicellularorganisms. However, as used herein, the term “microorganisms” alsoincludes viruses.

Preferably, the compositions of the invention comprise at least onecationic anti-microbial agent selected from anti-bacterial, anti-fungal,anti-algal, anti-sporal, anti-viral, anti-yeastal and anti-moldal agentsand mixtures thereof. More preferably, the compositions of the inventioncomprise at least one anti-bacterial, anti-viral, antifungal and/oranti-moldal agent.

As used herein, the terms anti-bacterial, anti-fungal, anti-algal,anti-viral, anti-yeastal and anti-moldal agents are intended to refer toagents, which inhibit the growth of the respective microorganisms but donot necessarily kill the microorganisms and agents which kill therespective microorganisms. Thus, for example, within the termanti-bacterial we include agents, which inhibit the growth of bacteriabut may not necessarily kill bacteria and bactericidal agents which dokill bacteria.

As the skilled person will appreciate, the word ending “cidal” as usedin for example “bactericidal” and “fungicidal” is used to describeagents which kill the microorganism to which it refers. Thus, in theseexamples, bactericidal refers to an agent that kills bacteria andfungicidal refers to an agent that kills fungus. Examples ofbactericides include myobactericides and tuberculocides. Preferably, thecompositions of the invention comprise at least one agent selected frombactericidal, fungicidal, algicidal, sporicidal, virucidal, yeasticidaland moldicidal agents and mixtures thereof. More preferably, thecompositions of the invention comprise at least one bactericidal,virucidal, fungicidal and/or moldicidal agent.

“Wet efficacy” is determined by a test process where a liquid suspensionof microorganisms is directly combined with a liquid mixture of achemical disinfectant. The liquid suspension of microorganisms can beadded directly to the liquid chemical or chemical can be added directlyto the suspension of microorganisms. Alternatively the microorganismsuspension may be dried onto a surface thereby creating a “carrier”. Thecarrier can then be added to a liquid mixture of chemistry or thechemistry can be added to the carrier so that in either casemicroorganisms are combined with a liquid solution of chemistry.Microbial reduction is measured by counting the number of survivingmicroorganisms following some time period after which disinfectant andmicroorganisms are combined. That assay is then mathematically convertedto a measurement of percent or log reduction of the test organisms.

“Dry efficacy” is determined by a test that measures the antimicrobialeffect of a chemical residue dried onto a surface. This takes the formof a chemical being applied to a clean sterilized surface or “carrier”.The film is allowed to dry or cure. The carrier is then optionallysubjected to simulated “wear” or “abrasion” to test the durability ofthe surface film. The final step is application of a liquidmicroorganism suspension to the surface of the dry carrier and assay forsurvivors following some exposure time. Microbial reduction is measuredby counting the number of surviving microorganisms following applicationof the suspension to the carrier surface. That assay is thenmathematically converted to a measurement of percent or log reduction ofthe test organisms.

The compositions of the invention are effective against a wide range oforganisms, including Gram negative and Gram positive spore formers,yeasts, and viruses.

By way of example, the microorganisms which the compositions of thepresent invention can be effective against include:

Viruses such as HIV-1 (AIDS Virus), Hepatitis B Virus (HVB), Hepatitis CVirus (HCV), Adenovirus, Herpes Simplex, Influenza (including seasonalflu, H1 N1 and H5N1), Respiratory Syncytial Virus (RSV), Vaccinia, AvianInfluenza virus, Avian Bronchitis, Pseudorabies virus, Canine Distemper,Newcastle Disease, Rubella, Avian Polyomavirus, Feline leukemia, Felinepicornavirus. Infectious Bovine rhinotracheitis, Infectious Bronchitis(Avian IBV)₁. Rabies, Transmissible gastroenteritis virus, Marek'sDisease.

Funguses such as Trichophyton mentagrophytes, Aspergillus niger, Candidaalbicans, Aspergillus flavus, Aspergillus fumigatus, Trichophytoninterdigitale, Alternaria tenius, Fusarium oxysporum, Geotrichumcandidum, Penicillium digitatum, Phytophthora infestans, Rhizopusnigricans, Trichoderma harzianum, Trichophyton interdigitale.

Bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus,Salmonella choleraesuis, Acinetobacter baumannii, Brevibacteriumammoniagenes, Campylobacter jejuni, Enterobacter aerogenes, Escherichiacoli, Klebsiella pneumoniae, Proteus mirabilis, Pseudomonas cepacia,Salmonella schottmuelleri, Salmonella typhi, Salmonella typhimurium,Serratia marcescens, Shigella dysenteriae, Shigella flexneri, Shigellasonnei, Staphyloccus epidermidis, Streptoccus faecalis, Streptoccusfaecalis (Vancomycin resistant), Streptococcus pyogenes, Vibrio chlorae,Xanthomonas axonopodis pv citri (Citrus canker), Acinetobactercalcoaceticus, Bordetella bronchiseptica, Chlamydia psittaci,Enterobacter cloacae, Enterococcus faecalis, Fusobacterium necrophorum,Legionella pneumophila, Listeria monocytogenes, Pasteurella multocida,Proteus vulgaris, Salmonella enteritidis, Mycoplasma gallisepticum,Yersinia enterocolitica, Aeromonas salmonicida, Pseudomonas putida,Vibrio anguillarum.

In particular, the compositions of the invention are effective againstP. aeruginosa (ATCC 15442, PaFH72/a), E. coli (ATCC 10536, ECFH64/a,0157:H7 (toxin producing strain), CCFRA/896, 0157:1-17 (non-toxigenicstrain), CCFAA/6896, ATCC 10538), S. aureus (including MRSA, (e.g. NCTC12493 MRSA, ATCC 12493 MRSA), VISA, ATCC 6538, 5a FH73/a), Entercoccushirea (ATCC 10541, EhFH 65/a), Feline Coronavirus (SARS surrogate).Feline Calcivirus (Hum. Norovirus surrogate), Salmonella typhimurium(StFH 68/b), Yersinia enterocolitica (YE FH67/b), Listeria monocytogenes(Lm FH66/c), Saccharomyces cerevisiae, Bacillus Subtilis (ATCC 6633),Bacillus stearothermophilus (NCTC 10339), Clostridium dificile (NCTC11209), Candida albicans (ATCC 1023), Aspergillus niger (ATCC 16404),Mycobacterium smegmatis (TB stimulant) and Influenza (including seasonalflu, H1 N1 and H5N1).

A further subject-matter of the present invention includes a process fordisinfecting and/or cleaning a hard surface. Dilutions for use solutionsare preferably within the range of a 1:16 dilution and a 1:256 dilution.

DETAILED DESCRIPTION OF THE INVENTION Definitions

So that the invention may be more readily understood certain terms arefirst defined.

“Cleaning” means to perform or aid in soil removal, bleaching, microbialpopulation reduction, or combination thereof.

As used herein, the term “ware” refers to items such as eating andcooking utensils and other hard surfaces such as showers, sinks,toilets, bathtubs, countertops, windows, mirrors, transportationvehicles, and floors. As used herein, the term “warewashing” refers towashing, cleaning, or rinsing ware.

As used herein, the term “hard surface” includes showers, sinks,toilets, bathtubs, countertops, windows, mirrors, transportationvehicles, floors, and the like.

As used herein, the phrase “health care surface” refers to a surface ofan instrument, a device, a cart, a cage, furniture, a structure, abuilding, or the like that is employed as part of a health careactivity. Examples of health care surfaces include surfaces of medicalor dental instruments, of medical or dental devices, of autoclaves andsterilizers, of electronic apparatus employed for monitoring patienthealth, and of floors, walls, or fixtures of structures in which healthcare occurs. Health care surfaces are found in hospital, surgical,infirmity, birthing, mortuary, and clinical diagnosis rooms. Thesesurfaces can be those typified as “hard surfaces” (such as walls,floors, bed-pans, etc.), or fabric surfaces, e.g., knit, woven, andnon-woven surfaces (such as surgical garments, draperies, bed linens,bandages, etc.), or patient-care equipment (such as respirators,diagnostic equipment, shunts, body scopes, wheel chairs, beds, etc.), orsurgical and diagnostic equipment. Health care surfaces include articlesand surfaces employed in animal health care.

As used herein, the term “instrument” refers to the various medical ordental instruments or devices that can benefit from cleaning using watertreated according to the methods of the present invention.

As used herein, the phrases “medical instrument,” “dental instrument,”“medical device,” “dental device,” “medical equipment,” or “dentalequipment” refer to instruments, devices, tools, appliances, apparatus,and equipment used in medicine or dentistry. Such instruments, devices,and equipment can be cold sterilized, soaked or washed and then heatsterilized, or otherwise benefit from cleaning using water treatedaccording to the present invention. These various instruments, devicesand equipment include, but are not limited to: diagnostic instruments,trays, pans, holders, racks, forceps, scissors, shears, saws (e.g. bonesaws and their blades), hemostats, knives, chisels, rongeurs, files,nippers, drills, drill bits, rasps, burrs, spreaders, breakers,elevators, clamps, needle holders, carriers, clips, hooks, gouges,curettes, retractors, straightener, punches, extractors, scoops,keratomes, spatulas, expressors, trocars, dilators, cages, glassware,tubing, catheters, cannulas, plugs, stents, scopes (e.g., endoscopes,stethoscopes, and arthoscopes) and related equipment, and the like, orcombinations thereof.

As used herein, “weight percent (wt-%),” “percent by weight,” “% byweight,” and the like are synonyms that refer to the concentration of asubstance as the weight of that substance divided by the total weight ofthe composition and multiplied by 100.

As used herein, the term “about” modifying the quantity of an ingredientin the compositions of the invention or employed in the methods of theinvention refers to variation in the numerical quantity that can occur,for example, through typical measuring and liquid handling proceduresused for making concentrates or use solutions in the real world; throughinadvertent error in these procedures; through differences in themanufacture, source, or purity of the ingredients employed to make thecompositions or carry out the methods; and the like. The term about alsoencompasses amounts that differ due to different equilibrium conditionsfor a composition resulting from a particular initial mixture. Whetheror not modified by the term “about,” the claims include equivalents tothe quantities

It should be noted that, as used in this specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the content clearly dictates otherwise. Thus, for example,reference to a composition containing “a compound” includes a mixture oftwo or more compounds. It should also be noted that the term “or” isgenerally employed in its sense including “and/or” unless the contentclearly dictates otherwise.

The recitation of numerical ranges by endpoints includes all numberssubsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3,3.80, 4, and 5).

In one aspect, the compositions of the invention must contain criticalratios of a quaternary ammonium compound, and a cationic anti-microbialcompound as well as a chelant and surfactant. The anti-microbialagent(s) used in the present invention are preferably water soluble atroom temperature and pressure.

Quaternary Ammonium Compounds

Quaternary ammonium compounds, also known as “quats”, typically compriseat least one quaternary ammonium cation with an appropriate anion. Thequaternary ammonium cations are permanently charged, independent of thepH of their solution. The structure of the cation can be represented asfollows:

The groups R₁, R₂, R₃ and R₄ can vary within wide limits and examples ofquaternary ammonium compounds that have anti-microbial properties willbe well known to the person of ordinary skill in the art.

Each group R₁, R₂, R₃ and R₄ may, for example, independently be asubstituted or unsubstituted and/or straight chain or branched and/orinterrupted or uninterrupted alkyl, aryl, alkylaryl, arylalkyl,cycloalkyl, (aromatic or non-aromatic) heterocyclyl or alkenyl group.Alternatively, two or more of R₁, R₂, R₃ and R₄ may together with thenitrogen atom form a substituted or unsubstituted heterocyclic ring. Thetotal number of carbon atoms in the groups R₁, R₂, R₃ and R₄ must be atleast 4. Typically the sum of the carbon atoms in the groups R₁, R₂, R₃and R₄ is 10 or more. In a preferred aspect of the invention at leastone of the groups R₁, R₂, R₃ and R₄ contains from 8 to 18 carbon atoms.For example, 1, 2, 3 or 4 of R₁, R₂, R₃ and R₄ can contain from 8 to 18carbon atoms or 10 to 16 carbon atoms.

Suitable substituents for the groups R-i, R₂, R₃ and R₄ may be selectedfrom the group consisting of alkyl, substituted alkyl, alkenyl,substituted alkenyl, heterocyclyl, substituted heterocyclyl, cycloalkyl,substituted cycloalkyl, aryl, substituted aryl, alkylaryl, substitutedalkylaryl, arylalkyl, substituted arylalkyl, F, Cl, Br, I, —OR¹, —NR¹R″,—CF₃, —CN, —NO₂, —C₂R¹, —SR′, —N₃, —C(═O)NR′R″, —NR¹C(O)R″,—C(═O)R\—C(═O)OR\—OC(O)R¹, —O(CR′R^(x))_(r)C(═O)R′,O(CR′R″)_(r)NR″C(O)R′, —O(CR′R″)_(r)NR″SO₂R′, —OC(O)NR¹R″, —NR¹C(O)OR″,—SO₂R″, —SO₂NR¹R″, and —NR¹SO₂R″, wherein R¹ and R″ are individuallyhydrogen, C₁-C₈ alkyl, cycloalkyl, heterocyclyl, aryl, or arylalkyl, andr is an integer from 1 to 6, or R¹ and R″ together form a cyclicfunctionality, wherein the term “substituted” as applied to alkyl,alkenyl, heterocyclyl, cycloalkyl, aryl, alkylaryl and arylalkyl refersto the substituents described above, starting with F and ending with—NR¹SO₂R″.

When one or more of R₁, R₂, R₃ and R₄ is interrupted, suitableinterrupting groups include but are not limited to heteroatoms such asoxygen, nitrogen, sulphur, and phosphorus-containing moieties (e.g.phosphinate). A preferred interrupting group is oxygen.

Suitable anions for the quats include but are not limited to halideanions such as chloride, fluoride, bromide or iodide and the non-halidesulphonate.

Preferred quats are those having the formula: (CH₃)_(n)(A)_(m)N⁺X—wherein A may be as defined above in relation to R₁, R₂, R₃ and R₄. X″is selected from chloride, fluoride, bromide or iodide and sulphonate(preferably chloride or bromide), n is from 1 to 3 (preferably 2 or 3)and m is from 1 to 3 (preferably 1 or 2) provided that the sum of n andm is 4. Preferably, A is a C₆₋₂₀ (e.g. C₈₋₁₈, i.e. having 8, 9, 10, 11,12, 13, 14, 15, 16, 17 or 18 carbon atoms or C₈₋₁₂ or C₁₂₋₁₈)substituted or unsubstituted and/or straight chain or branched and/orinterrupted or uninterrupted alkyl, aryl, alkylaryl, arylalkyl orcycloalkyl group (wherein suitable substituents are as defined above inrelation to R₁, R₂, R₃ and R₄). Each group A may be the same ordifferent.

A preferred group of the compounds of formula (CH₃)_(n)(A)_(m)N⁺X′ arethose wherein n=3 and m=1. In such compounds A may be as defined aboveand is preferably a C₆₋₂₀ substituted or unsubstituted and/or straightchain or branched and/or interrupted or uninterrupted alkyl, aryl, oralkylaryl group. Examples of this type of quaternary ammonium compoundinclude Cetrimide (which is predominately trimethyltetradecylammoniumbromide), dodecyltrimethylammonium bromide, trimethyltetradecylammoniumbromide, hexadecyltrimethylammonium bromide.

Another preferred group of the compounds of formula (CH₃)_(n)(A)_(m)N⁺X′are those wherein n=2 and m=2. In such compounds A may be as definedabove in relation to R₁, R₂, R₃ and R₄. Preferably A is a C₆₋₂₀substituted or unsubstituted and/or straight chain or branched and/orinterrupted or uninterrupted alkyl, aryl, or alkylaryl group. Forexample, A may represent a straight chain, unsubstituted anduninterrupted C₈₋₁₂ alkyl group or a benzyl group. In these compounds,the groups A may be the same or different. Examples of this type ofcompound include didecyl dimethyl ammonium chloride and dioctyl dimethylammonium chloride.

Examples of the preferred quaternary ammonium compounds described aboveinclude the group of compounds which are generally called benzalkoniumhalides and aryl ring substituted derivatives thereof. Examples ofcompounds of this type include benzalkonium chloride, which has thestructural formula:

wherein R may be as defined above in relation to R₁, R₂, R₃ and R₄.Preferably, R is a C₈₋₁₈ alkyl group or the benzalkonium chloride isprovided and/or used as a mixture of C₈₋is alkyl groups, particularly amixture of straight chain, unsubstituted and uninterrupted alkyl groupsn-C₈H₁₇ to n-C₁₈H₃7, e.g. n-Ci₂H₂5 to nC₁₈H₃₇ mainly n-C₁₂H₂s (dodecyl),n-C₁₄H₂₉ (tetradecyl), and n-C₁₆H₃₃ (hexadecyl).

Other preferred quaternary ammonium compounds include those in which thebenezene ring is substituted, for example alkyldimethyl ethylbenzylammonium chloride. As an example, a mixture containing, for example,equal molar amounts of alkyl dimethyl benzyl ammonium chloride andalkyldimethyl ethylbenzyl ammonium chloride may be used.

Other quaternary ammonium compounds suitable for use in the inventioninclude, but are not limited to, alkylpyridinium compounds, such ascetylpyridinium chloride, and bridged cyclic amino compounds such as thehexaminium compounds.

Other examples of quats which may be used in the present inventioninclude Cetalkonium Chloride, Cetylpyridinium Chloride, GlycidylTrimethyl Ammonium Chloride, Stearalkonium Chloride; Zephiran chloride(R); Hyamine 3500; Diisobutylphenoxyethoxyethyldimethylbenzylammoniumchloride: Hyamine 1622(R); Cetalkonium Chloride;Cetyldimethylbenzyl-ammonium chloride; Triton K 12;Cetyltrimethylammonium bromide; Retarder LA; 1-Hexadecylpyridiniumchloride; Glycidyltrimethyl-ammonium chloride: Benzethonium Chloride CAS121-54-0; Cetalkonium Chloride CAS 122-18-9: Cetrimide CAS 8044-71-1;Cetylpyridinium Chloride (anhydrous) CAS 123-03-5; StearalkoniumChloride CAS 122-19-0; Cetrimonium Bromide CAS 57-09-0.

Particularly preferred quaternary ammonium compounds includebenzyldimethyl-n-tetradecyl-ammonium chloride,benzyldimethyl-n-dodecyl-ammonium chloride,n-dodecyl-n-tetradecyldimethyl-ammonium chloride andbenzyl-Ci2-C₁₆-alkyl-dimethyl-ammonium chloride,benzyl-cocoalkyl-dimethyl-ammonium chloride, di-n-decyldimethylammoniumchloride.

The compositions of the invention include a synergistic combination of acationic anti-microbial, a chelant and an amine oxide surfactant incombination with the quaternary ammonium compound.

According to the invention the applicants have discovered that a usesolution composition in the critical range of about 195 to about 7800ppm of a single quaternary ammonium compound, about 19.5 to about 1250ppm of cationic biocide provides a film forming solution that retainsits antimicrobial activity and remains on the surface after numerousabrasion cycles. In additional preferred embodiments the use compositionalso includes from about 62.5-2000 ppm amine oxide surfactant and 62.5to 2000 ppm chelant.

On an actives weight basis, the invention comprises a ratio of cationicbiocide to single quaternary ammonium compound to of about 1:10 to about1:0.5 More preferred is a range of about 1:6.0 to about 1:1.0 and mostpreferred is a ratio of about 1:4.0 to about 1:2.0.

In a preferred embodiment the ratio of cationic biocide to one or morequaternary ammonium is less than 1:1 (i.e. less than 50% by weight ofcationic biocide. In a more preferred embodiment the ratio is from about0.99 to about 0.01 cationic biocide to about 1 of quaternary ammonium onan actives weight basis. These compositions of the invention comprise atleast one cationic biocide, but they preferably do not contain more thana single class of quaternary ammonium compound

Thus Applicants have found that in order to keep efficacy, the ratio ofquaternary ammonium to surfactant should be from about 2:1 to about 7:1respectively.

Applicants have also found that the composition preferably includes achelant. Again here. Applicants have found that the chelant should be ina ration of from about 2:1 to about 3.5 to 1 of quaternary ammonium tochelant respectively. In a preferred embodiment the chelant isethylenediaminetetraacetic acid.

Cationic Biocide

The cationic biocide agent is that component of the composition providesat least part of the biocidal/antimicrobial activity. That is, thecationic biocide agent has at least some biocidal/antimicrobial activityfor at least one microorganism. It is generally considered the mainactive component of the compositions described herein. The cationicbiocide agent includes an effective amount of one or more biocide agentsselected from the group consisting of biguanides and bisbiguanides suchas chlorhexidine and its various salts including but not limited to thedigluconate, diacetate, dimethosulfate, and dilactate salts, as well ascombinations thereof; polymeric quaternary ammonium compounds such aspolyhexamethylenebiguanide; small molecule quaternary ammonium compoundssuch as benzalkonium halides; and compatible combinations thereof. It isparticularly important, however, with cationic biocide agents in a saltform to use a counter ion that ensures solubility in aqueous fluid abovethe minimum inhibitory concentration (MIC) of the treatment organism. Ifthe solubility limit is less than the MIC, treatment may be ineffective.

The classes of cationic biocide agent suitable in the present inventionare discussed further below.

Biguanides

This class of biocides is represented by the formula:

R—NH—C(NH)—NH—C(NH)—NH(CH₂)_(n)NHC(NH)—NH—C(NH)—NH—R

Where n=3-10, preferably 4-8, and most preferably 6; and R is C₄-C₁₈branched or straight chain alkyl optionally substituted in availablepositions by halogen or C₆-C₁₂ aryl or alkaryl optionally substituted inavailable positions by halogen.

The preferred compound of this class is chlorhexidine. This may bepresent as the free base but is preferably present as a disalt ofacetate, gluconate, lactate, methosulfate (CH₃OSO₃ ⁻), or a halide orcombinations thereof. The most preferred compound is chlorhexidinedigluconate (CHG). Other anions may be useful. Many salts ofchlorhexidine have high solubility (>1 g/100 mL) in alcohol/watersystems and are therefore useful in compositions of this invention.

The biocides of this class are particularly preferred in formulationsthat are aqueous and protected from light. This is believed to reducethe degradation of the compound. Care must also be taken whenformulating chlorhexidine as well as other cationic biocide compounds toavoid inactivation by sequestering it in micelles which may be formed byincorporation of surfactants and/or emulsifiers. Preferred compositionsof this invention are essentially free of surfactants and/oremulsifiers.

Bis(biguanide)s such as chlorhexidine are very basic and capable offorming multiple ionic bonds with anionic materials. For this reason,biguanide-containing compositions are preferably free of anioniccompounds that can result in precipitation of the biocide. Anionicsurfactants useful, for example, as wetting agents, may also need to beavoided. Halide salts may need to be avoided. For example, chlorhexidinedigluconate (CHG) will precipitate rapidly in the presence of halidesalts above a concentration of about 0.1M. Therefore, if a systemincludes CHG or other biocide of this class, and needs to comprise saltsfor stability or other purposes, preferably gluconate salts such astriethanolamine gluconate or sodium gluconate, are used.

It has been found that in use compositions of the invention whichcomprise the two components of a quaternary ammonium compound and acationic biocide in for example the ratios set out above have anadvantageous anti-microbial effect. For example, such compositions canhave an enhanced kill rate when they are applied to a surface (so called“wet kill”) and/or they can also have a residual anti-microbial effectin that they control, reduce or prevent the formation of new microbialcolonies at the surface (so called “dry kill”) and/or they are effectiveat significantly lower concentration of anti-microbial agent thanpreviously known compositions.

Surfactants

In some embodiments, the compositions of the present invention include asurfactant. Surfactants suitable for use with the compositions of thepresent invention include, but are not limited to, semi-polar nonionicsurfactants such as amine oxides. In addition, other surfactants such asanionic surfactants, and zwitterionic surfactants may be used. In someembodiments, the compositions of the present invention include about 0.4wt % to about 12.8 wt % of a surfactant. In some embodiments, thecompositions of the present invention include about 62.5 ppm to about2000 ppm of a surfactant.

Semi-Polar Nonionic Surfactants

The semi-polar type of nonionic surface active agents are the preferredclass of surfactants useful in compositions of the present invention.Semi-polar nonionic surfactants include the amine oxides, phosphineoxides, sulfoxides and their alkoxylated derivatives. Most preferred areamine oxide surfactants of am R¹ chain length of 8.

Amine oxides are tertiary amine oxides corresponding to the generalformula:

wherein the arrow is a conventional representation of a semi-polar bond;and, R¹, R², and R³ may be aliphatic, aromatic, heterocyclic, alicyclic,or combinations thereof. Generally, for amine oxides of detergentinterest, R¹ is an alkyl radical of from about 8 to about 24 carbonatoms; R² and R³ are alkyl or hydroxyalkyl of 1-3 carbon atoms or amixture thereof; R² and R³ can be attached to each other, e.g. throughan oxygen or nitrogen atom, to form a ring structure; R⁴ is an alkyleneor a hydroxyalkylene group containing 2 to 3 carbon atoms; and n rangesfrom 0 to about 20. An amine oxide can be generated from thecorresponding amine and an oxidizing agent, such as hydrogen peroxide.

Useful water soluble amine oxide surfactants are selected from theoctyl, decyl, dodecyl, isododecyl, coconut, or tallow alkyl di-(loweralkyl) amine oxides, specific examples of which are octyldimethylamineoxide, nonyldimethylamine oxide, decyldimethylamine oxide,undecyldimethylamine oxide, dodecyldimethylamine oxide,iso-dodecyldimethyl amine oxide, tridecyldimethylamine oxide,tetradecyldimethylamine oxide, pentadecyldimethylamine oxide,hexadecyldimethylamine oxide, heptadecyldimethylamine oxide,octadecyldimethylaine oxide, dodecyldipropylamine oxide,tetradecyldipropylamine oxide, hexadecyldipropylamine oxide,tetradecyldibutylamine oxide, octadecyldibutylamine oxide,bis(2-hydroxyethyl)dodecylamine oxide,bis(2-hydroxyethyl)-3-dodecoxy-1-hydroxypropylamine oxide,dimethyl-(2-hydroxydodecyl)amine oxide, 3,6,9-trioctadecyldimethylamineoxide and 3-dodecoxy-2-hydroxypropyldi-(2-hydroxyethyl)amine oxide.

The compositions of the invention may optionally include additionalsurfactants such as the following.

Nonionic Surfactants

Suitable additional nonionic surfactants for use with the compositionsof the present invention include alkoxylated surfactants. Suitablealkoxylated surfactants include EO/PO copolymers, capped EO/POcopolymers, alcohol alkoxylates, capped alcohol alkoxylates, mixturesthereof, or the like. Suitable alkoxylated surfactants for use assolvents include EO/PO block copolymers, such as the Pluronic andreverse Pluronic surfactants; alcohol alkoxylates, such as Dehypon LS-54(R-(EO)₅(PO)₄) and Dehypon LS-36 (R-(EO)₃(PO)₆); and capped alcoholalkoxylates, such as Plurafac LF221 and Tegoten EC11; mixtures thereof,or the like.

Anionic Surfactants

Anionic sulfate surfactants suitable for use in the present compositionsinclude alkyl ether sulfates, alkyl sulfates, the linear and branchedprimary and secondary alkyl sulfates, alkyl ethoxysulfates, fatty oleylglycerol sulfates, alkyl phenol ethylene oxide ether sulfates, theC₅-C₁₇ acyl-N—(C₁-C₄ alkyl) and —N—(C₁-C₂ hydroxyalkyl) glucaminesulfates, and sulfates of alkylpolysaccharides such as the sulfates ofalkylpolyglucoside, and the like. Also included are the alkyl sulfates,alkyl poly(ethyleneoxy) ether sulfates and aromatic poly(ethyleneoxy)sulfates such as the sulfates or condensation products of ethylene oxideand nonyl phenol (usually having 1 to 6 oxyethylene groups permolecule).

Anionic sulfonate surfactants suitable for use in the presentcompositions also include alkyl sulfonates, the linear and branchedprimary and secondary alkyl sulfonates, and the aromatic sulfonates withor without substituents.

Anionic carboxylate surfactants suitable for use in the presentcompositions include carboxylic acids (and salts), such as alkanoicacids (and alkanoates), ester carboxylic acids (e.g. alkyl succinates),ether carboxylic acids, and the like. Such carboxylates include alkylethoxy carboxylates, alkyl aryl ethoxy carboxylates, alkyl polyethoxypolycarboxylate surfactants and soaps (e.g. alkyl carboxyls). Secondarycarboxylates useful in the present compositions include those whichcontain a carboxyl unit connected to a secondary carbon. The secondarycarbon can be in a ring structure, e.g. as in p-octyl benzoic acid, oras in alkyl-substituted cyclohexyl carboxylates. The secondarycarboxylate surfactants typically contain no ether linkages, no esterlinkages and no hydroxyl groups. Further, they typically lack nitrogenatoms in the head-group (amphiphilic portion). Suitable secondary soapsurfactants typically contain 11-13 total carbon atoms, although morecarbons atoms (e.g., up to 16) can be present. Suitable carboxylatesalso include acylamino acids (and salts), such as acylgluamates, acylpeptides, sarcosinates (e.g. N-acyl sarcosinates), taurates (e.g. N-acyltaurates and fatty acid amides of methyl tauride), and the like.

Suitable anionic surfactants include alkyl or alkylaryl ethoxycarboxylates of the following formula:

R—O—(CH₂CH₂O)_(n)(CH₂)_(m)—CO₂X  (3)

in which R is a C₈ to C₂₂ alkyl group or

in which R¹ is a C₄-C₁₆ alkyl group; n is an integer of 1-20; m is aninteger of 1-3; and X is a counter ion, such as hydrogen, sodium,potassium, lithium, ammonium, or an amine salt such as monoethanolamine,diethanolamine or triethanolamine. In some embodiments, n is an integerof 4 to 10 and m is 1. In some embodiments, R is a C₈-C₁₆ alkyl group.In some embodiments, R is a C₁₂-C₁₄ alkyl group, n is 4, and m is 1.

In other embodiments, R is

and R¹ is a C₆-C₁₂ alkyl group. In still yet other embodiments, R¹ is aC₉ alkyl group, n is 10 and m is 1.

Such alkyl and alkylaryl ethoxy carboxylates are commercially available.These ethoxy carboxylates are typically available as the acid forms,which can be readily converted to the anionic or salt form. Commerciallyavailable carboxylates include, Neodox 23-4, a C₁₂₋₁₃ alkyl polyethoxy(4) carboxylic acid (Shell Chemical), and Emcol CNP-110, a C₉ alkylarylpolyethoxy (10) carboxylic acid (Witco Chemical). Carboxylates are alsoavailable from Clariant, e.g. the product Sandopan® DTC, a C₁₃ alkylpolyethoxy (7) carboxylic acid.

Amphoteric Surfactants

Amphoteric, or ampholytic, surfactants contain both a basic and anacidic hydrophilic group and an organic hydrophobic group. These ionicentities may be any of anionic or cationic groups described herein forother types of surfactants. A basic nitrogen and an acidic carboxylategroup are the typical functional groups employed as the basic and acidichydrophilic groups. In a few surfactants, sulfonate, sulfate,phosphonate or phosphate provide the negative charge.

Amphoteric surfactants can be broadly described as derivatives ofaliphatic secondary and tertiary amines, in which the aliphatic radicalmay be straight chain or branched and wherein one of the aliphaticsubstituents contains from about 8 to 18 carbon atoms and one containsan anionic water solubilizing group, e.g., carboxy, sulfo, sulfato,phosphato, or phosphono. Amphoteric surfactants are subdivided into twomajor classes known to those of skill in the art and described in“Surfactant Encyclopedia” Cosmetics & Toiletries, Vol. 104 (2) 69-71(1989). The first class includes acyl/dialkyl ethylenediaminederivatives (e.g. 2-alkyl hydroxyethyl imidazoline derivatives) andtheir salts. The second class includes N-alkylamino acids and theirsalts. Some amphoteric surfactants can be envisioned as fitting intoboth classes.

Amphoteric surfactants can be synthesized by methods known to those ofskill in the art. For example, 2-alkyl hydroxyethyl imidazoline issynthesized by condensation and ring closure of a long chain carboxylicacid (or a derivative) with dialkyl ethylenediamine. Commercialamphoteric surfactants are derivatized by subsequent hydrolysis andring-opening of the imidazoline ring by alkylation—for example withchloroacetic acid or ethyl acetate. During alkylation, one or twocarboxy-alkyl groups react to form a tertiary amine and an ether linkagewith differing alkylating agents yielding different tertiary amines.

Long chain imidazole derivatives having application in the presentinvention generally have the general formula:

wherein R is an acyclic hydrophobic group containing from about 8 to 18carbon atoms and M is a cation to neutralize the charge of the anion,generally sodium. Commercially prominent imidazoline-derived amphotericsthat can be employed in the present compositions include for example:Cocoamphopropionate, Cocoamphocarboxy-propionate, Cocoamphoglycinate,Cocoamphocarboxy-glycinate, Cocoamphopropyl-sulfonate, andCocoamphocarboxy-propionic acid. Amphocarboxylic acids can be producedfrom fatty imidazolines in which the dicarboxylic acid functionality ofthe amphodicarboxylic acid is diacetic acid and/or dipropionic acid.

The carboxymethylated compounds (glycinates) described herein abovefrequently are called betaines. Betaines are a special class ofamphoteric discussed herein below in the section entitled, ZwitterionSurfactants.

Long chain N-alkylamino acids are readily prepared by reaction RNH₂, inwhich R=C₈-C₁₈ straight or branched chain alkyl, fatty amines withhalogenated carboxylic acids.

Alkylation of the primary amino groups of an amino acid leads tosecondary and tertiary amines. Alkyl substituents may have additionalamino groups that provide more than one reactive nitrogen center. Mostcommercial N-alkylamine acids are alkyl derivatives of beta-alanine orbeta-N(2-carboxyethyl) alanine Examples of commercial N-alkylamino acidampholytes having application in this invention include alkyl beta-aminodipropionates, RN(C₂H₄COOM)₂ and RNHC₂H₄COOM. In an embodiment, R can bean acyclic hydrophobic group containing from about 8 to about 18 carbonatoms, and M is a cation to neutralize the charge of the anion.

Suitable amphoteric surfactants include those derived from coconutproducts such as coconut oil or coconut fatty acid. Additional suitablecoconut derived surfactants include as part of their structure anethylenediamine moiety, an alkanolamide moiety, an amino acid moiety,e.g., glycine, or a combination thereof; and an aliphatic substituent offrom about 8 to 18 (e.g., 12) carbon atoms. Such a surfactant can alsobe considered an alkyl amphodicarboxylic acid. These amphotericsurfactants can include chemical structures represented as:C₁₂-alkyl-C(O)—NH—CH₂—CH₂—N⁺(CH₂—CH₂—CO₂Na)₂—CH₂—CH₂—OH orC₁₂-alkyl-C(O)—N(H)—CH₂—CH₂—N⁺(CH₂—CO₂Na)₂—CH₂—CH₂—OH. Disodiumcocoampho dipropionate is one suitable amphoteric surfactant and iscommercially available under the tradename Miranol™ FBS from RhodiaInc., Cranbury, N.J. Another suitable coconut derived amphotericsurfactant with the chemical name disodium cocoampho diacetate is soldunder the tradename Mirataine™ JCHA, also from Rhodia Inc., Cranbury,N.J.

A typical listing of amphoteric classes, and species of thesesurfactants, is given in U.S. Pat. No. 3,929,678 issued to Laughlin andHeuring on Dec. 30, 1975. Further examples are given in “Surface ActiveAgents and Detergents” (Vol. I and II by Schwartz, Perry and Berch).

Zwitterionic Surfactants

Zwitterionic surfactants can be thought of as a subset of the amphotericsurfactants and can include an anionic charge. Zwitterionic surfactantscan be broadly described as derivatives of secondary and tertiaryamines, derivatives of heterocyclic secondary and tertiary amines, orderivatives of quaternary ammonium, quaternary phosphonium or tertiarysulfonium compounds. Typically, a zwitterionic surfactant includes apositive charged quaternary ammonium or, in some cases, a sulfonium orphosphonium ion; a negative charged carboxyl group; and an alkyl group.Zwitterionics generally contain cationic and anionic groups which ionizeto a nearly equal degree in the isoelectric region of the molecule andwhich can develop strong “inner-salt” attraction betweenpositive-negative charge centers. Examples of such zwitterionicsynthetic surfactants include derivatives of aliphatic quaternaryammonium, phosphonium, and sulfonium compounds, in which the aliphaticradicals can be straight chain or branched, and wherein one of thealiphatic substituents contains from 8 to 18 carbon atoms and onecontains an anionic water solubilizing group, e.g., carboxy, sulfonate,sulfate, phosphate, or phosphonate. Betaine and sultaine surfactants areexemplary zwitterionic surfactants for use herein.

A general formula for these compounds is:

wherein R¹ contains an alkyl, alkenyl, or hydroxyalkyl radical of from 8to 18 carbon atoms having from 0 to 10 ethylene oxide moieties and from0 to 1 glyceryl moiety; Y is selected from the group consisting ofnitrogen, phosphorus, and sulfur atoms; R² is an alkyl or monohydroxyalkyl group containing 1 to 3 carbon atoms; x is 1 when Y is a sulfuratom and 2 when Y is a nitrogen or phosphorus atom, R³ is an alkylene orhydroxy alkylene or hydroxy alkylene of from 1 to 4 carbon atoms and Zis a radical selected from the group consisting of carboxylate,sulfonate, sulfate, phosphonate, and phosphate groups.

Examples of zwitterionic surfactants having the structures listed aboveinclude:4-[N,N-di(2-hydroxyethyl)-N-octadecylammonio]-butane-1-carboxylate;5-[S-3-hydroxypropyl-S-hexadecylsulfonio]-3-hydroxypentane-1-sulfate;3-[P,P-diethyl-P-3,6,9-trioxatetracosanephosphonio]-2-hydroxypropane-1-phosphate;3-[N,N-dipropyl-N-3-dodecoxy-2-hydroxypropyl-ammonio]-propane-1-phosphonate;3-(N,N-dimethyl-N-hexadecylammonio)-propane-1-sulfonate;3-(N,N-dimethyl-N-hexadecylammonio)-2-hydroxy-propane-1-sulfonate;4-[N,N-di(2(2-hydroxyethyl)-N(2-hydroxydodecyl)ammonio]-butane-1-carboxylate;3-[S-ethyl-S-(3-dodecoxy-2-hydroxypropyl)sulfonio]-propane-1-phosphate;3-[P,P-dimethyl-P-dodecylphosphonio]-propane-1-phosphonate; and S[N,N-di(3-hydroxypropyl)-N-hexadecylammonio]-2-hydroxy-pentane-1-sulfate.The alkyl groups contained in said detergent surfactants can be straightor branched and saturated or unsaturated.

The zwitterionic surfactant suitable for use in the present compositionsincludes a betaine of the general structure:

These surfactant betaines typically do not exhibit strong cationic oranionic characters at pH extremes nor do they show reduced watersolubility in their isoelectric range. Unlike “external” quaternaryammonium salts, betaines are compatible with anionics. Examples ofsuitable betaines include coconut acylamidopropyldimethyl betaine;hexadecyl dimethyl betaine; C₁₂₋₁₄ acylamidopropylbetaine; C₈₋₁₄acylamidohexyldiethyl betaine; 4-C₁₄₋₁₆acylmethylamidodiethylammonio-1-carboxybutane; C₁₆₋₁₈acylamidodimethylbetaine; C₁₂₋₁₆ acylamidopentanediethylbetaine; andC₁₂₋₁₆ acylmethylamidodimethylbetaine.

Sultaines useful in the present invention include those compounds havingthe formula (R(R¹)₂N⁺R²SO³⁻, in which R is a C₆-C₁₈ hydrocarbyl group,each R¹ is typically independently C₁-C₃ alkyl, e.g. methyl, and R² is aC₁-C₆ hydrocarbyl group, e.g. a C₁-C₃ alkylene or hydroxyalkylene group.

A typical listing of zwitterionic classes, and species of thesesurfactants, is given in U.S. Pat. No. 3,929,678 issued to Laughlin andHeuring on Dec. 30, 1975. Further examples are given in “Surface ActiveAgents and Detergents” (Vol. I and II by Schwartz, Perry and Berch).

Chelants

The compositions of the invention can also include a chelant at a levelof from 0.4 wt. % to 12.8 wt. %, or 62.5 ppm to about 2000 ppm in a usecomposition. Chelation herein means the binding or complexation of a bi-or multidentate ligand. These ligands, which are often organiccompounds, are called chelants, chelators, chelating agents, and/orsequestering agent. Chelating agents form multiple bonds with a singlemetal ion. Chelants, are chemicals that form soluble, complex moleculeswith certain metal ions, inactivating the ions so that they cannotnormally react with other elements or ions to produce precipitates. Theligand forms a chelate complex with the substrate. The term is reservedfor complexes in which the metal ion is bound to two or more atoms ofthe chelant.

Suitable chelating agents can be selected from the group consisting ofamino carboxylates, amino phosphonates, polyfunctionally-substitutedaromatic chelating agents and mixtures thereof. Preferred chelants foruse herein are amino carboxylates.

Amino carboxylates include ethylenediaminetetra-acetates,N-hydroxyethylethylenediaminetriacetates, nitrilo-triacetates,ethylenediamine tetrapro-prionates, triethylenetetraaminehexacetates,diethylenetriaminepentaacetates, and ethanoldi-glycines, alkali metal,ammonium, and substituted ammonium salts therein and mixtures therein.As well as MGDA (methyl-glycine-diacetic acid), and salts andderivatives thereof and GLDA (glutamic-N,N-diacetic acid) and salts andderivatives thereof. GLDA (salts and derivatives thereof) is especiallypreferred according to the invention, with the tetrasodium salt thereofbeing especially preferred.

Other suitable chelants include amino acid based compound or a succinatebased compound. The term “succinate based compound” and “succinic acidbased compound” are used interchangeably herein. Other suitable chelantsare described in U.S. Pat. No. 6,426,229. Particular suitable chelantsinclude; for example, aspartic acid-N-monoacetic acid (ASMA), asparticacid-N,N-diacetic acid (ASDA), aspartic acid-N-monopropionic acid(ASMP), iminodisuccinic acid (IDS), Imino diacetic acid (IDA),N-(2-sulfomethyl)aspartic acid (SMAS), N-(2-sulfoethyl)aspartic acid(SEAS), N-(2-sulfomethyl)glutamic acid (SMGL), N-(2-sulfoethyl)glutamicacid (SEGL), N-methyliminodiacetic acid (MIDA),.quadrature.-alanine-N,N-diacetic acid (.quadrature.-ALDA),serine-N,N-diacetic acid (SEDA), isoserine-N,N-diacetic acid (ISDA),phenylalanine-N,N-diacetic acid (PHDA), anthranilic acid-N,N-diaceticacid (ANDA), sulfanilic acid-N,N-diacetic acid (SLDA),taurine-N,N-diacetic acid (TUDA) and sulfomethyl-N,N-diacetic acid(SMDA) and alkali metal salts or ammonium salts thereof. Also suitableis ethylenediamine disuccinate (“EDDS”), especially the [S,S] isomer asdescribed in U.S. Pat. No. 4,704,233. Furthermore,Hydroxyethyleneiminodiacetic acid, Hydroxyiminodisuccinic acid,Hydroxyethylene diaminetriacetic acid is also suitable.

Other chelants include homopolymers and copolymers of polycarboxylicacids and their partially or completely neutralized salts, monomericpolycarboxylic acids and hydroxycarboxylic acids and their salts.Preferred salts of the abovementioned compounds are the ammonium and/oralkali metal salts, i.e. the lithium, sodium, and potassium salts, andparticularly preferred salts are the sodium salts.

Suitable polycarboxylic acids are acyclic, alicyclic, heterocyclic andaromatic carboxylic acids, in which case they contain at least twocarboxyl groups which are in each case separated from one another by,preferably, no more than two carbon atoms. Polycarboxylates whichcomprise two carboxyl groups include, for example, water-soluble saltsof, malonic acid, (ethyl enedioxy) diacetic acid, maleic acid,diglycolic acid, tartaric acid, tartronic acid and fumaric acid.Polycarboxylates which contain three carboxyl groups include, forexample, water-soluble citrate. Correspondingly, a suitablehydroxycarboxylic acid is, for example, citric acid. Another suitablepolycarboxylic acid is the homopolymer of acrylic acid. Preferred arethe polycarboxylates end capped with sulfonates.

Amino phosphonates are also suitable for use as chelating agents andinclude ethylenediaminetetrakis(methylenephosphonates) as DEQUEST.Preferred, these amino phosphonates that do not contain alkyl or alkenylgroups with more than about 6 carbon atoms.

Polyfunctionally-substituted aromatic chelating agents are also usefulin the compositions herein such as described in U.S. Pat. No. 3,812,044.Preferred compounds of this type in acid form aredihydroxydisulfobenzenes such as 1,2-dihydroxy-3,5-disulfobenzene.

Further suitable polycarboxylates chelants for use herein include citricacid, lactic acid, acetic acid, succinic acid, formic acid allpreferably in the form of a water-soluble salt. Other suitablepolycarboxylates are oxodisuccinates, carboxymethyloxysuccinate andmixtures of tartrate monosuccinic and tartrate disuccinic acid such asdescribed in U.S. Pat. No. 4,663,071.

Other Additional Ingredients

Additional ingredients that may be used in the formulations of theinvention include but are not limited to water, antioxidants,thickeners, foam makers/boosters such as alkanolamides and abrasives,sequestrants such as nitrilotriacetic acid (NTA), tetrasodium EDTA,other acetic acid derivatives and mixtures thereof; salts such as sodiumchloride and citrate salts, pH modifiers, for example acids such ascitric, sulfamic, hydrochloric, phosphoric, nitric, lactic, formic,acetic or gluconic acids or other mineral or organic acids or bases suchas sodium or potassium hydroxide and mono-, di- or tri-ethanoiamine,colorants, fragrances, emollients and hair and/or skin rejuvenatingand/or protecting agents.

In some embodiments, the compositions of the present invention caninclude other additional ingredients. Additional ingredients suitablefor use with the compositions of the present invention include, but arenot limited to, acidulants, stabilizing agents, buffers, detergents,wetting agents, defoaming agents, thickeners, aesthetic enhancing agents(i.e., colorants, odorants, or perfumes) and other cleaning agents.These additional ingredients can be preformulated with the compositionsof the invention or added to the system before, after, or substantiallysimultaneously with the addition of the compositions of the presentinvention. Additionally, the compositions can be used in conjunctionwith one or more conventional cleaning agents.

Acidulants

In some embodiments, the compositions of the present invention includean acidulant. The acidulant can act as a catalyst for conversion ofcarboxylic acid to peroxycarboxylic acid. The acidulant can be effectiveto form a concentrate composition with pH of about 1 or less. Theacidulant can be effective to form a use composition with pH of about 5,about 5 or less, about 4, about 4 or less, about 3, about 3 or less,about 2, about 2 or less, or the like. In some embodiments, an acidulantcan be used to lower the pH of an alkaline cleaning solution to a pH ofabout 10, about 10 or less, about 9, about 9 or less, about 8, about 8or less, about 7, about 7 or less, about 6, or about 6 or less. In anembodiment, the acidulant includes an inorganic acid. Suitable inorganicacids include, but are not limited to, sulfuric acid, sodium bisulfate,phosphoric acid, nitric acid, hydrochloric acid. In some embodiments,the acidulant includes an organic acid. Suitable organic acids include,but are not limited to, methane sulfonic acid, ethane sulfonic acid,propane sulfonic acid, butane sulfonic acid, xylene sulfonic acid,benzene sulfonic acid, formic acid, acetic acid, mono, di, ortri-halocarboyxlic acids, picolinic acid, dipicolinic acid, and mixturesthereof. In some embodiments, the compositions of the present inventionare free or substantially free of a phosphorous based acid.

In some embodiments, acidulant selected can also function as astabilizing agent. Thus, the compositions of the present invention canbe substantially free of an additional stabilizing agent.

Stabilizing Agents

In some embodiments, the compositions of the present invention includeone or more stabilizing agents in addition to the chelant componentmentioned supra. The stabilizing agents can be used, for example, tostabilize the composition components and prevent their interaction.

Suitable stabilizing agents include, for example, chelating agents orsequestrants. Suitable sequestrants include, but are not limited to,organic chelating compounds that sequester metal ions in solution,particularly transition metal ions. Such sequestrants include organicamino- or hydroxy-polyphosphonic acid complexing agents (either in acidor soluble salt forms), carboxylic acids (e.g., polymericpolycarboxylate), hydroxycarboxylic acids, aminocarboxylic acids, orheterocyclic carboxylic acids, e.g., pyridine-2,6-dicarboxylic acid(dipicolinic acid).

In some embodiments, the compositions of the present invention includedipicolinic acid as a stabilizing agent. Compositions includingdipicolinic acid can be formulated to be free or substantially free ofphosphorous. It has also been observed that the inclusion of dipicolinicacid in a composition of the present invention aids in achieving thephase stability of the compositions, compared to other conventionalstabilizing agents, e.g., 1-hydroxy ethylidene-1,1-diphosphonic acid(CH₃C(PO₃H₂)₂OH) (HEDP).

In other embodiments, the sequestrant can be or include phosphonic acidor phosphonate salt. Suitable phosphonic acids and phosphonate saltsinclude HEDP; ethylenediamine tetrakis methylenephosphonic acid (EDTMP);diethylenetriamine pentakis methylenephosphonic acid (DTPMP);cyclohexane-1,2-tetramethylene phosphonic acid; amino[tri(methylenephosphonic acid)]; (ethylene diamine[tetra methylene-phosphonic acid)];2-phosphene butane-1,2,4-tricarboxylic acid; or salts thereof, such asthe alkali metal salts, ammonium salts, or alkyloyl amine salts, such asmono, di, or tetra-ethanolamine salts; picolinic, dipicolinic acid ormixtures thereof. In some embodiments, organic phosphonates, e.g., HEDPare included in the compositions of the present invention.

Commercially available food additive chelating agents includephosphonates sold under the trade name DEQUEST® including, for example,1-hydroxyethylidene-1,1-diphosphonic acid, available from MonsantoIndustrial Chemicals Co., St. Louis, Mo., as DEQUEST® 2010;amino(tri(methylenephosphonic acid)), (N[CH₂PO₃H₂]₃), available fromMonsanto as DEQUEST® 2000; ethylenediamine[tetra(methylenephosphonicacid)] available from Monsanto as DEQUEST® 2041; and2-phosphonobutane-1,2,4-tricarboxylic acid available from Mobay ChemicalCorporation, Inorganic Chemicals Division, Pittsburgh, Pa., as BayhibitAM.

The sequestrant can be or include aminocarboxylic acid type sequestrant.Suitable aminocarboxylic acid type sequestrants include the acids oralkali metal salts thereof, e.g., amino acetates and salts thereof.Suitable aminocarboxylates include N-hydroxyethylaminodiacetic acid;hydroxyethylenediaminetetraacetic acid, nitrilotriacetic acid (NTA);ethylenediaminetetraacetic acid (EDTA);N-hydroxyethyl-ethylenediaminetriacetic acid (HEDTA);diethylenetriaminepentaacetic acid (DTPA); and alanine-N,N-diaceticacid; and the like; and mixtures thereof.

The sequestrant can be or include a polycarboxylate. Suitablepolycarboxylates include, for example, polyacrylic acid, maleic/olefincopolymer, acrylic/maleic copolymer, polymethacrylic acid, acrylicacid-methacrylic acid copolymers, hydrolyzed polyacrylamide, hydrolyzedpolymethacrylamide, hydrolyzed polyamide-methacrylamide copolymers,hydrolyzed polyacrylonitrile, hydrolyzed polymethacrylonitrile,hydrolyzed acrylonitrile-methacrylonitrile copolymers, polymaleic acid,polyfumaric acid, copolymers of acrylic and itaconic acid, phosphinopolycarboxylate, acid or salt forms thereof, mixtures thereof, and thelike.

Wetting or Defoaming Agents

Also useful in the compositions of the invention are wetting anddefoaming agents. Wetting agents function to increase the surfacecontact or penetration activity of the antimicrobial composition of theinvention. Wetting agents which can be used in the composition of theinvention include any of those constituents known within the art toraise the surface activity of the composition of the invention.

Generally, defoamers which can be used in accordance with the inventioninclude silica and silicones; aliphatic acids or esters; alcohols;sulfates or sulfonates; amines or amides; halogenated compounds such asfluorochlorohydrocarbons; vegetable oils, waxes, mineral oils as well astheir sulfonated or sulfated derivatives; fatty acids and/or their soapssuch as alkali, alkaline earth metal soaps; and phosphates and phosphateesters such as alkyl and alkaline diphosphates, and tributyl phosphatesamong others; and mixtures thereof.

In some embodiments, the compositions of the present invention caninclude antifoaming agents or defoamers which are of food grade qualitygiven the application of the method of the invention. To this end, oneof the more effective antifoaming agents includes silicones. Siliconessuch as dimethyl silicone, glycol polysiloxane, methylphenolpolysiloxane, trialkyl or tetralkyl silanes, hydrophobic silicadefoamers and mixtures thereof can all be used in defoamingapplications. Commercial defoamers commonly available include siliconessuch as Ardefoam® from Armour Industrial Chemical Company which is asilicone bound in an organic emulsion; Foam Kill® or Kresseo® availablefrom Krusable Chemical Company which are silicone and non-silicone typedefoamers as well as silicone esters; and Anti-Foam A® and DC-200 fromDow Corning Corporation which are both food grade type silicones amongothers.

Thickening or Gelling Agents

The compositions of the present invention can include any of a varietyof known thickeners. Suitable thickeners include natural gums such asxanthan gum, guar gum, or other gums from plant mucilage; polysaccharidebased thickeners, such as alginates, starches, and cellulosic polymers(e.g., carboxymethyl cellulose); polyacrylates thickeners; andhydrocolloid thickeners, such as pectin. In an embodiment, the thickenerdoes not leave contaminating residue on the surface of an object. Forexample, the thickeners or gelling agents can be compatible with food orother sensitive products in contact areas. Generally, the concentrationof thickener employed in the present compositions or methods will bedictated by the desired viscosity within the final composition.

Solidification Agent

The present compositions can include a solidification agent, which canparticipate in maintaining the compositions in a solid form. In someembodiments, the solidification agent can form and/or maintain thecomposition as a solid. In other embodiments, the solidification agentcan solidify the composition without unacceptably detracting from theeventual release of the sulfonated peroxycarboxylic acid. Thesolidification agent can include, for example, an organic or inorganicsolid compound having a neutral inert character or making a functional,stabilizing or detersive contribution to the present composition.Suitable solidification agents include solid polyethylene glycol (PEG),solid polypropylene glycol, solid EO/PO block copolymer, amide, urea(also known as carbamide), nonionic surfactant (which can be employedwith a coupler), anionic surfactant, starch that has been madewater-soluble (e.g., through an acid or alkaline treatment process),cellulose that has been made water-soluble, inorganic agent, poly(maleicanhydride/methyl vinyl ether), polymethacrylic acid, other generallyfunctional or inert materials with high melting points, mixturesthereof, and the like;

Suitable glycol solidification agents include a solid polyethyleneglycol or a solid polypropylene glycol, which can, for example, havemolecular weight of about 1,400 to about 30,000. In certain embodiments,the solidification agent includes or is solid PEG, for example PEG 1500up to PEG 20,000. In certain embodiments, the PEG includes PEG 1450, PEG3350, PEG 4500, PEG 8000, PEG 20,000, and the like. Suitable solidpolyethylene glycols are commercially available from Union Carbide underthe tradename CARBOWAX.

Suitable amide solidification agents include stearic monoethanolamide,lauric diethanolamide, stearic diethanolamide, stearic monoethanolamide, cocodiethylene amide, an alkylamide, mixtures thereof, and thelike. In an embodiment, the present composition can include glycol(e.g., PEG) and amide.

Suitable nonionic surfactant solidification agents include nonylphenolethoxylate, linear alkyl alcohol ethoxylate, ethylene oxide/propyleneoxide block copolymer, mixtures thereof, or the like. Suitable ethyleneoxide/propylene oxide block copolymers include those sold under thePluronic tradename (e.g., Pluronic 108 and Pluronic F68) andcommercially available from BASF Corporation. In some embodiments, thenonionic surfactant can be selected to be solid at room temperature orthe temperature at which the composition will be stored or used. Inother embodiments, the nonionic surfactant can be selected to havereduced aqueous solubility in combination with the coupling agent.Suitable couplers that can be employed with the nonionic surfactantsolidification agent include propylene glycol, polyethylene glycol,mixtures thereof, or the like.

Suitable anionic surfactant solidification agents include linear alkylbenzene sulfonate, alcohol sulfate, alcohol ether sulfate, alpha olefinsulfonate, mixtures thereof, and the like. In an embodiment, the anionicsurfactant solidification agent is or includes linear alkyl benzenesulfonate. In an embodiment, the anionic surfactant can be selected tobe solid at room temperature or the temperature at which the compositionwill be stored or used.

Suitable inorganic solidification agents include phosphate salt (e.g.,alkali metal phosphate), sulfate salt (e.g., magnesium sulfate, sodiumsulfate or sodium bisulfate), acetate salt (e.g., anhydrous sodiumacetate), Borates (e.g., sodium borate), Silicates (e.g., theprecipitated or fumed forms (e.g., Sipernat 50® available from Degussa),carbonate salt (e.g., calcium carbonate or carbonate hydrate), otherknown hydratable compounds, mixtures thereof, and the like. In anembodiment, the inorganic solidification agent can include organicphosphonate compound and carbonate salt, such as an E-Form composition.

In some embodiments, the compositions of the present invention caninclude any agent or combination of agents that provide a requisitedegree of solidification and aqueous solubility can be included in thepresent compositions. In other embodiments, increasing the concentrationof the solidification agent in the present composition can tend toincrease the hardness of the composition. In yet other embodiments,decreasing the concentration of solidification agent can tend to loosenor soften the concentrate composition.

In some embodiments, the solidification agent can include any organic orinorganic compound that imparts a solid character to and/or controls thesoluble character of the present composition, for example, when placedin an aqueous environment. For example, a solidifying agent can providecontrolled dispensing if it has greater aqueous solubility compared toother ingredients in the composition. Urea can be one suchsolidification agent. By way of further example, for systems that canbenefit from less aqueous solubility or a slower rate of dissolution, anorganic nonionic or amide hardening agent may be appropriate.

In some embodiments, the compositions of the present invention caninclude a solidification agent that provides for convenient processingor manufacture of the present composition. For example, thesolidification agent can be selected to form a composition that canharden to a solid form under ambient temperatures of about 30 to about50° C. after mixing ceases and the mixture is dispensed from the mixingsystem, within about 1 minute to about 3 hours, or about 2 minutes toabout 2 hours, or about 5 minutes to about 1 hour.

The compositions of the present invention can include solidificationagent at any effective amount. The amount of solidification agentincluded in the present composition can vary according to the type ofcomposition, the ingredients of the composition, the intended use of thecomposition, the quantity of dispensing solution applied to the solidcomposition over time during use, the temperature of the dispensingsolution, the hardness of the dispensing solution, the physical size ofthe solid composition, the concentration of the other ingredients, theconcentration of the cleaning agent in the composition, and other likefactors. Suitable amounts can include about 1 to about 99 wt-%, about1.5 to about 85 wt-%, about 2 to about 80 wt-%, about 10 to about 45wt-%, about 15% to about 40 wt-%, about 20% to about 30 wt-%, about 30%to about 70%, about 40% to about 60%, up to about 50 wt-%, about 40% toabout 50%

Use Compositions

The compositions of the present invention include concentratecompositions and use compositions. For example, a concentratecomposition can be diluted, for example with water, to form a usecomposition. In an embodiment, a concentrate composition can be dilutedto a use solution before to application to an object. For reasons ofeconomics, the concentrate can be marketed and an end user can dilutethe concentrate with water or an aqueous diluent to a use solution.

The level of active components in the concentrate composition isdependent on the intended dilution factor and the desired activity ofthe antimicrobial composition. Generally, a dilution of about 1 fluidounce to about 10 gallons of water to about 10 fluid ounces to about 1gallon of water is used for aqueous compositions of the presentinvention. In some embodiments, higher use dilutions can be employed ifelevated use temperature (greater than 25° C.) or extended exposure time(greater than 30 seconds) can be employed. In the typical use locus, theconcentrate is diluted with a major proportion of water using commonlyavailable tap or service water mixing the materials at a dilution ratioof about 3 to about 40 ounces of concentrate per 100 gallons of water.

In certain embodiments, a use composition can include about 0.01 toabout 10 wt-% of a concentrate composition and about 90 to about 99.99wt-% diluent; or about 0.1 to about 1 wt-% of a concentrate compositionand about 99 to about 99.9 wt-% diluent.

Amounts of an ingredient in a use composition can be calculated from theamounts listed herein for concentrate compositions and these dilutionfactors. The concentrated compositions of the present invention aregenerally diluted such that the quaternary ammonium compound is presentat from about 195 ppm to about 7800 ppm and the cationic biocide ispresent from about 19.5 to 1250. It is to be understood that all valuesand ranges between these values and ranges are encompassed by thepresent invention.

The compositions of the invention are resistant to touching and generalabrasion. This means that the compositions of the invention provide aresidual anti-microbial effect even when the surface is touched, rubbedor abraded as would be typical during normal interaction between asurface and individuals working on or around that surface. Thedurability of the antimicrobial film is not intended to be permanent butit can be replenished by wiping the treated surface with a saturatedcloth, mop, sponge or other suitable delivery mechanism. The compositioncan also be applied by spraying or flooding the surface with thebiocidal composition. The synergistic combination of the componentsincreases durability of the film, making it more resistant to wear andabrasion.

It will be appreciated that the actual concentration of components in acomposition of the invention will depend on the intended use of thatcomposition. For disinfecting uses, such as cleaning of hospital wardsand equipment to help prevent the spread of disease such as MRSA, higherconcentrations are required than for certain sanitizing applications.

In use the compositions of the invention act to substantially reduce orcontrol the formation of microbial colonies on or at the surface towhich they are applied. This means that not only do the compositions ofthe invention kill any microorganisms that are present on a surface whenthey are applied to that surface (so called “wet kill”), they also havea residual effect in that they prevent the formation of new microbialcolonies at the surface (so called “dry kill”).

The present compositions provide improved durability, i.e. thecompositions of the invention remain on the surface and prevent thegrowth of colonies of microorganisms. The residual effect can often beseen even after a treated surface has been touched or abraded numeroustimes.

Anti-microbial compositions are considered to have residual efficacy if,in the residual efficacy test described herein, they give a reduction inthe number of microorganisms which is at least log 3.0. Preferably ananti-microbial composition having a residual effect and tested in thismanner will give a log reduction of at least about 3.0, more preferablyat least about 4.0 and most preferably about 6.0 or more, up to totalkill or substantially total kill (zero survivors) under the testconditions described above.

In a particular aspect, the present invention provides anti-microbialcompositions which have residual efficacy. In other words thesecompositions, when tested in accordance with the residual efficacy testdescribed herein have an anti-microbial efficacy within the parameterset out in the paragraph above.

The formulations of the present invention typically comprise ananti-microbial composition as described above in combination withcompatible ingredients which allow the formulation to perform itsprimary purpose.

In particular, the present invention provides formulations comprising anantimicrobial compositions suitable for a variety of consumerapplications. Examples of the formulations of the invention include, butare not limited to, surface cleaners such as those intended for use inbathrooms, kitchens, living areas, hard floor cleaners, carpet cleaners;furniture cleaners; glass/mirror cleaners; toilet care productsincluding solid toilet cleaners such as rim devices and those designedto be placed in the cistern, liquid toilet cleaners excluding thosecomprising hypochlorite bleaches; dishwashing products such as washingup liquids and preparations from dishwashing machines such asdishwashing solids (e.g. powders and tablets) & liquids; laundryproducts such as solid detergents (e.g. powders and tablets), liquiddetergents and fabric conditioners and “2 in 1” products comprisingdetergent and fabric conditioner; cleaning products intended for useoutdoors such as those for cleaning for wood, stone, concrete orplastics, for example patio cleaner, garden furniturecleaners/treatments, BBQ cleaners, wall and fence cleaners/treatments,plant sprays such as those intended to remove insects such as aphidesfrom plants; food sprays, such as those suitable for use in foodpreservation; personal care products such as bath and shower products;soaps, including liquid and solid soaps, hand sanitizers, deodorants andantiperspirants, hair care products including shampoos, for exampleanti-scalp odor shampoos, shampoos for the control of head lice eggs andanti-dandruff shampoos, hair conditioners, hair styling products such ashair mousses, gels and sprays, skin care products such as shavingproducts, cosmetics and products for hair removal; baby productsincluding baby cleaning and cleansing products such as baby bath, soaps,wipes, moisturizers, nappy rash cream, products for cleaning surfacesthat have regular & high incidence of infant & baby contact; first aidproducts and products for treating ailments and illnesses, includingproducts for the topical treatment and/or prevention of minor infectionssuch as athletes foot, spot/acne prevention/treatment products; foothygiene products, including those for use on the foot and those for thetreatment/deodorization of foot ware, particularly sports foot wear;products for cleaning and/or deodorizing vehicles such as cars.

The formulations of the invention comprise an anti-microbial compositionas described above. The pH of the formulations of the invention can varywithin wide limits. Typically, the pH of a formulation of the inventionwill be similar to that of known formulations which are intended to beused for the same purpose or a similar purpose to a given formulation ofthe invention. For example The pH can range from 4 to 11 depending onthe dilution, and specific use of the composition.

Examples of formulations of the invention are as follows:Below is a use composition of the invention.

Preferred More Preferred Most preferred Quaternary  195-7800 ppm390-3906 ppm 780-1953 ppm ammonium Cationic 19.5-1250 ppm 87.5-937.5ppm   156-625 ppm biocide Amine Oxide 62.5-2000 ppm 125-1500 ppm250-1000 ppm Chelant 62.5-2000 ppm 125-1500 ppm 250-1000 ppmThe following is a concentrate composition that can be diluted at a 1:64ratio.

Preferred More Preferred Most preferred Quaternary  1.25-50%  2.5-25% 5-12.5% ammonium Cationic 0.125-8.0%  0.56-6.0%   1.0-4% Amine Oxide0.4-12.8% 0.8-9.6% 1.6-6.4% Chelant 0.4-12.8% 0.8-9.6% 1.6-6.4%

Typically, the anti-microbial composition is incorporated into a simpleconventional detergent solution or added to a “final rinse” duringcleaning.

According to a further aspect of the invention, there is provided theuse of an antimicrobial composition of the invention to prevent theformation of colonies of microorganisms on a surface at which it isprovided.

According to yet a further aspect of the invention, there is providedthe use of a formulation to prevent the formation of colonies ofmicroorganisms on a surface at which it is provided.

The invention also provides a process for making the compositions of theinvention. The process comprises the steps of (A) mixing at least partof quaternary ammonium compound and adding the cationic biocidecomponent in the appropriate amounts to achieve the synergy of the two,and any further components such as chelants, and surfactants, andagitating the resulting mixture until a clear solution is formed.

Typically, the process to produce the compositions of the invention iscarried out at room temperature with stirring.

The present invention provides compositions obtainable by the processset out above. The compositions of the invention may be prepared in aconcentrated form (i.e. with little or no polar solvent) and dilutedwith water when used.

The following are non-limiting examples of the invention and areintended for purposes of illustration only.

EXAMPLES Example 1 Hard Surface Efficacy of PHMB/Quaternary AmmoniumChloride Blend Vs. Stabilized Chlorine Dioxide

The objective of this analysis was to examine the efficacy of variouscompositions against Staphylococcus aureus ATCC 6538 and Klebsiellapneumoniae ATCC 4352 after application to inanimate, non-porous,non-food contact surfaces.

Test Method: Residual Self-Sanitizing Activity on Hard, Non-PorousSurfaces

-   -   Test Surface Preparation:    -   Clean glass test surfaces were decontaminated by immersing in        reagent alcohol and allowing to air dry at room temperature. The        test surfaces were dried in sterile glass petri dishes        containing 2 layers of Whatman No. 2 paper. Each petri dish had        two surfaces in them and were dried for 1 day in a biological        safety cabinet with the lids cracked open.    -   Test Surface Inoculation and Coating:    -   The initial inoculum suspension was prepared by making 10⁻² and        10⁻⁴ dilutions from a 48-54 hour broth culture of the test        system. The 10⁻⁴ dilution was allowed to stand for 15 minutes        before 10 μL was spread evenly over the entire coupon. The        coupons were dried at 35±2° C. for 30 minutes with the lids        cracked open. 50 μL of the diluted test substances of 0.01%        Triton X 100 (for controls) was gently spread over the surface        of the coupons (2 coupons per treatment). The coupons were dried        overnight at room temperature in a biological safety cabinet        with the lids closed. The initial inoculum suspension was        enumerated by plating serial dilutions in duplicate.    -   Preparation of the Inocula:    -   The reinoculation suspension was prepared by making 10⁻² and        10⁻⁴ dilutions from an 18-24 hour broth culture of the test        system. 5 mL of the 10⁻⁴ dilution was added to 5 mL of Phosphate        Buffered Dilution Water (PBDW). A 5% Fetal Bovine Serum (FBS)        soil was added to the inoculum suspension, vortexted, and        allowed to stand for 15 minutes. The final inoculum suspension        was prepared by making 10⁻¹ dilution from an 18-24 hour culture        of the test system, adding 5% FBS, vortexted, and allowed to        stand for 15 minutes. Both inoculum suspensions were serially        diluted and pour plated in duplicate. The suspensions were not        allowed to stand with organic soil for longer than 8 hours.    -   Operating Technique:    -   10 wears were performed using the Gardner Abrasion Tester. The        wears alternated between dry or wet, beginning with a dry wear        and ending with a wet wear. The abrasion boat was assembled in        the following manner for each wear: a thin foam pad and a cotton        strip were wrapped around the flat surface of the boat. For dry        wears, the boat was run over two coupons for one cycle. For wet        wears, the boat was sprayed for 1 second with sterile water (in        a Prevail sprayer) at a distance of about 75 cm before it was        run over two coupons for one cycle. The coupons were        reinoculated with 10 μL of the reinoculation suspension, spread        evenly and dried at room temperature for 15 to 30 minutes.        Coupons were reinoculated after the first 5 wears. Approximately        10 minutes elapsed after the previous wear before the coupons        were reinoculated. After the final wear, 10 μL of the final        inoculum suspension was spread over the surface of each coupon.        At the end of the exposure time of 5 minutes, the coupons were        neutralized in 30 mL of D/E Broth in a centrifuge tube. The        centrifuge tubes were sonicated for 20 seconds and vortexed        vigorously for 1 minute. The tubes were serially diluted and        pour plated. As a neutralizer screen, an uninoculated slide was        coated with test substance as described in Test Surface        Inoculation and Coating and 0.2 mL of a 10² to 10³ CFU/mL of        test system was added. As a control for the neutralizer screen,        an uncoated slide added to the neutralizer and inoculated with        test system. The neutralizer screen sat for 30 minutes before        pour plating 1 mL and 0.1 mL of the neutralizer

Method Parameters:

-   -   Test Substances: Shield Medicare Products:        -   Test composition A (Quat/Biguanide a composition of the            invention)        -   Test composition B (Stabilized Chlorine Dioxide/Quat)        -   Biocide A is a ready to use solution of Biguanide and quat            on an actives basis of 2000 ppm PHMB and 5000 ppm QAC (ratio            of ˜1:2.5) It would fall within the intended ranges of the            invention        -   Triton X-100 A commercially available nonionic detergent            from Sigma Chemical, St. Louis Mo. with no antimicrobial            activity(control at 0.01%)    -   Test Systems: Staphylococcus aureus ATCC 6538        -   Klebsiella pneumoniae ATCC 4352    -   Organic Soil: 5% Fetal Bovine Serum    -   Test Surface: 1″×1″ Glass Surfaces        -   1 mm thick non-frosted microscope slides, cut into squares    -   Exposure Time: 5 minutes    -   Neutralizer Medium: 30 mL D/E Broth        -   A neutralizer screen showed that this neutralizer provided            adequate neutralization and was not detrimental to the test            systems.    -   Plating Medium: Tryptone Glucose Extract Agar    -   Incubation: 35° C. for 48 hours

Inoculum Numbers Culture Average Test System Description A B CFU/mLStaphylococcus Initial Inoculum 48 × 10³ 53 × 10³ 5.0 × 10⁴ aureusRe-inoculum 17 × 10³ 31 × 10³ 2.4 × 10⁴ ATCC 6538 Final Inoculum 50 ×10⁶ 43 × 10⁶ 4.6 × 10⁷

Staphylococcus aureus ATCC 6538 Average Log of Test Survivors (CFU/Individual Geometric Percent System Test Surface) Survivors Mean*Reduction Test <30  X₁: <1.48 <1.5 × 10²  >99.948 Composition <30  X₂:<1.48 A 3.7 × 10³ X₃: 3.57 Test 1.1 × 10⁵ X₁: 2.26 3.9 × 10³ 98.655Composition 1.4 × 10² X₂: 2.48 B X₃: 1.95 X₄: 1.48 TritonX 3.0 × 10⁵ X₁:5.48 2.9 × 10⁵ N/A 2.0 × 10⁵ X₂: 5.30 3.6 × 10⁵ X₃: 5.56 3.3 × 10⁵ X₄:5.52 [Antilog(X₁ + X₂)]/2.

Inoculum Numbers Culture Average Test System Description A B CFU/mLKlebsiella Inoculum 47 × 10³ 62 × 10³ 5.4 × 10⁴ pneumoniae Re-inoculum31 × 10³ 30 × 10³ 3.0 × 10⁴ ATCC 4352 Final Inoculum 39 × 10⁶ 48 × 10⁶4.4 × 10⁷

Klebsiella pneumoniae ATCC 4352 Average Log of Test Survivors (CFU/Individual Geometric Percent Substance Test Surface) Survivors Mean*Reduction Test 7.5 × 10¹ X₁: 1.88 8.4 × 10¹ >99.958 composition 4.5 ×10¹ X₂: 1.65 A <30  X₃: <1.48 4.8 × 10² X₄: 2.68 Test 1.0 × 10⁵ X₁: 5.002.2 × 10⁴ 89.000 Composition 7.1 × 10¹ X₂: 1.85 B 1.4 × 10⁵ X₃: 5.15 2.2× 10⁵ X₄: 5.34 TritonX 1.6 × 10⁵ X₁: 5.20 2.0 × 10⁵ N/A 2.6 × 10⁵ X₂:5.41 2.7 × 10⁵ X₃: 5.43 1.5 × 10⁵ X₄: 5.18 [Antilog(X₁ + X₂)]/2.

Conclusion:

To be defined as a sanitizer, the test substances on the hard inanimatesurface must reduce the total number of organisms by at least 99.9%(based on the Geometric Mean) on the surface within a 5 minute period(after the final inoculation).

Against S. aureus and K. pneumoniae, Test Composition A achieved therequired minimum percent reduction on the hard inanimate surface withinthe specified exposure time, and Test Composition B did not.

Example 2 Residual Self-Sanitizing Screen of Test Composition a

The objective of the analysis was to examine the residualself-sanitizing efficacy of against Staphylococcus aureus ATCC 6538 andKlebsiella pneumoniae ATCC 4352 after application to inanimate,non-porous, non-food contact surfaces.

Test Method: Residual Self-Sanitizing Activity on Hard, Non-PorousSurfaces Test System Preparation

-   1. At least three consecutive loop transfers of a 24 hour culture of    Staphylococcus aureus ATCC 6538 and Klebsiella pneumoniae ATCC 4352    were performed in 10 mL of AOAC Synthetic broth or AOAC Nutrient    broth (respectively) and incubated at 35° C.-   2. Initial Inoculum Suspension:    -   2.1 Vortex a 48-54 hour culture for 3-4 seconds.    -   2.2 Make two 1/100 dilutions in sterile PBDW and let stand for        15±1 minutes.-   3. Reinoculation Suspension:    -   3.1 Vortex an 18-24 hour culture for 3-4 seconds.    -   3.2 Make two 1/100 dilutions in sterile PBDW and on final        dilution of 5.0 mL of diluted culture in 5.0 mL of sterile PBDW.    -   3.3 Add organic coil load to equal 5%, vortex, and let stand for        15±1 minutes.-   4. Final Inoculum Suspension:    -   4.1 Vortex an 18-24 hour culture for 3-4 seconds.    -   4.2 Make one 1/10 dilution in sterile PBDW and vortex.    -   4.3 Add organic soil load to equal 5%, vortex, and let stand for        15±1 minutes.

Test Surface Preparation

-   1. Clean glass surfaces by rinsing in alcohol, then sterile water,    and allow to air dry.-   2. Decontaminate glass surfaces by immersing in absolute ethanol and    allowing to air dry (slides can be autoclaved if necessary).-   3. Transfer to individual glass petri dishes lined with 1-2 layers    of sterile Whatman No. 2 paper, and allow all surfaces to dry    completely prior to use (approximately one day).-   4. Inoculation of Test Surfaces    -   4.1. Apply a 10 μL aliquot of the Initial Inoculum Suspension        (Step 2 in the Test System Preparation) to each of 2-4 prepared        test surfaces, per test substance, per test system. Also        inoculate 2-4 prepared test surfaces, per test system, to be        used as control surfaces.    -   4.2. Spread inoculum to within ⅛ inch of the edge with the        pipette tip.    -   4.3. Dry with lids cracked at 35° C. for 30-35 minutes, or until        visibly dry.    -   4.4. Apply the test substance to the test surfaces on a clean        dry surface. Apply test substance to each test surface        appropriate to the application instructions. If no application        method is specified, apply 50 μL of the test substance to the        test surface and spread, with a sterile disposable loop, in an        even layer over entire test surface. Allow the surfaces to dry        overnight, covered, at room temperature.-   5. Apply a 0.01% TritonX 100 solution (made and filter sterilized on    the day of application) to each of the control surfaces in the same    manner as the test substances. Allow the control surfaces to dry    under the same conditions as the test surfaces.

Operating Technique

-   1. Wear and Reinoculation of Test and Control Surfaces: The treated    surfaces will undergo a wear and reinoculation regimen, which will    take place over at least a 24 hour period at room temperature.-   2. GardCo Washability and Wear Tester: A cycle equals one pass to    the left, and a return pass to the right. One pass on the abrasion    tester should provide a contact time with the surfaces of    approximately 2 seconds.-   3. Place one set of surfaces (two test or control surfaces) into the    cut out region of the surface wear area on the abrasion tester, and    perform one cycle of surface wears.-   4. Decontaminate the surface wear area with absolute ethanol between    each set of surface wears to prevent carryover contamination. Allow    the alcohol to completely evaporate before proceeding. Replace the    foam liner and the cotton cloth, on the abrasion boat assembly,    between each set of surface wears.-   5. Alternate dry-wears and wet-wears. For wet-wears, spray clean    cotton cloth with sterile distilled water, using a Preval sprayer,    from a distance of 75±1 cm for not more than one second. Immediately    attach the moistened abrasion boat assembly to the abrasion tester,    and perform one cycle of surface wears.-   6. Wait at least 15 minutes after each wear to reinoculate surfaces.    Reinoculate test and control surfaces by applying 10 μL of the    Reinoculation Suspension (Step 3 in Test System Preparation) and    spread the inoculum to within ⅛ inch of the surface edge with a the    pipette tip. Dry at least 30 minutes at room temperature before    proceeding with the next wear.-   7. The period between test substance application and the initiation    of the sanitizer test (carriers into neutralizer broth) must be at    least 24 hours.-   Enumeration of Survivors-   1. With the Final Inoculum Suspension (Step 4 in Test System    Preparation), inoculate the first surface with 10 μL, at time zero.    Begin inoculation about 5 seconds before time zero, and spread    aliquot over the surface so it is completed at time zero. Begin the    inoculation of the second surface similarly, at given intervals,    until all test and control surfaces have been inoculated.-   2. At 5 minutes (or other appropriate time) use alcohol-flamed    forceps to transfer the surfaces to 30 mL of neutralizer broth in a    50 mL centrifuge tube. Repeat until all test and control surfaces    have been completed.-   3. Sonicate the samples for 20±2 seconds in a sonicating water bath.    Then agitate the samples on an orbital shaker for 3-4 minutes at 250    rpm.-   4. Serially dilute the control sample suspensions in PBDW and    prepare duplicate pour plates of the 10⁻², 10⁻³ and 10⁻⁴ dilutions.    The control plates must have a minimum of 1×10⁴ CFU/mL for a valid    test.-   5. Serially dilute the test sample suspensions in PBDW and prepare    duplicate pour plates of the 10⁰, 10⁻² and 10⁻⁴ dilutions.-   6. Plate all samples within 30 minutes of their transfer to    neutralizer broth.

Method Parameters: Test Substance Formula:

Test composition A Raw Material Manufacturer Concentration Bardac 2250(50% active Lonza 1.0% Quaternary ammonium compound) Vantocil P (20%Biguanide) Arch Chemicals 1.0% Distilled Water  98%

-   -   Test Systems: Staphylococcus aureus ATCC 6538        -   Klebsiella pneumoniae ATCC 4352    -   Soil Challenge: 5% Bovine Serum    -   Test Material: 1″×1″ Glass Surfaces        -   1 mm thick non-frosted microscope slides, cut into squares    -   Test Substance Application: 3 sprays applied with a sprayer    -   Initial Inoculum Application Time: 2:30 pm (9-8-08)    -   Test Substance Application Time: 2:45 pm (9-8-08)    -   Final Inoculum Application Time: 3:05 pm (9-9-08)    -   Neutralizer Medium: 30 mL Dey Engley Broth        -   A neutralizer screen was performed as part of the testing,            verified that the neutralizer adequately neutralized the            product and was not detrimental to the tested organisms.    -   Plating Medium: Tryptone Glucose Extract Agar    -   Incubation: 35° C. for 48 hours

Inoculum Numbers Inoculum Average Test System Suspension A B CFU/mLStaphylococcus Initial 113 × 10³ 84 × 10³ 9.8 × 10⁴ aureus Reinoculation 61 × 10³ 53 × 10³ 5.7 × 10⁴ ATCC 6538 Final 139 × 10⁶ 132 × 10⁶  1.4 ×10⁸ Klebsiella Initial  81 × 10³ 62 × 10³ 7.2 × 10⁴ pneumoniaeReinoculation  60 × 10³ 54 × 10³ 5.7 × 10⁴ ATCC 4352 Final 100 × 10⁶ 120× 10⁷  1.1 × 10⁸

Staphylococcus aureus ATCC 6538 Test Survivors Survivors Average PercentSubstance (CFU/mL) (CFU/Carrier)* CFU/Carrier Reduction TritonX  96 ×10², 105 × 10² 2.9 × 10⁵, 3.2 × 10⁵ 3.9 × 10⁵ N/A 119 × 10², 200 × 10²3.6 × 10⁵, 6.0 × 10⁵ Test 0 × 10⁰, 0 × 10⁰ <30, <30, <30, <30<30 >99.992 Composition A 0 × 10⁰, 0 × 10⁰ *CFU/mL × 30

Klebsiella pneumoniae ATCC 4352 Test Survivors Survivors Average PercentSubstance (CFU/mL) (CFU/Carrier)* CFU/Carrier Reduction TritonX 149 ×10², 153 × 10² 4.5 × 10⁵, 4.6 × 10⁵  4.5 × 10⁵ N/A 152 × 10², 148 × 10²4.6 × 10⁵, 4.4 × 10⁵ Test 2 × 10⁰, 2 × 10⁰ 6.0 × 10¹, 6.0 × 10¹ <4.5 ×10¹ >99.990 Composition A 0 × 10⁰, 0 × 10⁰ <30, <30 *CFU/mL × 30

Conclusion:

To be defined as a sanitizer, the test substances on the hard inanimatesurface must reduce the total number of organisms by at least 99.9%(based on the Geometric Mean) on the surface within a 5 minute period(after the final inoculation).

Test composition A passed the residual hard surface sanitizer screenwith a 5 minute final exposure, against S. aureus and K. pneumoniae.

Example 3 Residual Self-Sanitizing Screen of Test Composition C

The objective of the analysis was to examine the residualself-sanitizing efficacy of Test composition C (a composition accordingto the invention) against Staphylococcus aureus ATCC 6538 and Klebsiellapneumoniae ATCC 4352 after application to inanimate, non-porous,non-food contact surfaces.

Test Method: Residual Self-Sanitizing Activity on Hard, Non-PorousSurfaces Test System Preparation

-   1. At least three consecutive loop transfers of a 24 hour culture of    Staphylococcus aureus ATCC 6538 and Klebsiella pneumoniae ATCC 4352    were performed in 10 mL of AOAC Synthetic broth or AOAC Nutrient    broth (respectively) and incubated at 35° C.-   2. Initial Inoculum Suspension:    -   2.1 Vortex a 48-54 hour culture for 3-4 seconds.    -   2.2 Make two 1/100 dilutions in sterile PBDW and let stand for        15±1 minutes.-   3. Reinoculation Suspension:    -   3.1 Vortex an 18-24 hour culture for 3-4 seconds.    -   3.2 Make two 1/100 dilutions in sterile PBDW and on final        dilution of 5.0 mL of diluted culture in 5.0 mL of sterile PBDW.    -   3.3 Add organic coil load to equal 5%, vortex, and let stand for        15±1 minutes.-   4. Final Inoculum Suspension:    -   4.1 Vortex an 18-24 hour culture for 3-4 seconds.    -   4.2 Make one 1/10 dilution in sterile PBDW and vortex.    -   4.3 Add organic soil load to equal 5%, vortex, and let stand for        15±1 minutes.

Test Surface Preparation

-   1. Clean glass surfaces by rinsing in alcohol, then sterile water,    and allow to air dry.-   2. Decontaminate glass surfaces by immersing in absolute ethanol and    allowing to air dry (slides can be autoclaved if necessary).-   3. Transfer to individual glass petri dishes lined with 1-2 layers    of sterile Whatman No. 2 paper, and allow all surfaces to dry    completely prior to use (approximately one day).-   4. Inoculation of Test Surfaces    -   4.1. Apply a 10 μL aliquot of the Initial Inoculum Suspension        (Step 2 in the Test System Preparation) to each of 2-4 prepared        test surfaces, per test substance, per test system. Also        inoculate 2-4 prepared test surfaces, per test system, to be        used as control surfaces.    -   4.2. Spread inoculum to within ⅛ inch of the edge with the        pipette tip.    -   4.3. Dry with lids cracked at 35° C. for 30-35 minutes, or until        visibly dry.    -   4.4. Apply the test substance to the test surfaces on a clean        dry surface. Apply test substance to each test surface        appropriate to the application instructions. If no application        method is specified, apply 50 μL of the test substance to the        test surface and spread, with a sterile disposable loop, in an        even layer over entire test surface. Allow the surfaces to dry        overnight, covered, at room temperature.-   5. Apply a 0.01% TritonX 100 solution (made and filter sterilized on    the day of application) to each of the control surfaces in the same    manner as the test substances. Allow the control surfaces to dry    under the same conditions as the test surfaces.

Operating Technique

-   1. Wear and Reinoculation of Test and Control Surfaces: The treated    surfaces will undergo a wear and reinoculation regimen, which will    take place over at least a 24 hour period at room temperature.-   2. GardCo Washability and Wear Tester: A cycle equals one pass to    the left, and a return pass to the right. One pass on the abrasion    tester should provide a contact time with the surfaces of    approximately 2 seconds.-   3. Place one set of surfaces (two test or control surfaces) into the    cut out region of the surface wear area on the abrasion tester, and    perform one cycle of surface wears.-   4. Decontaminate the surface wear area with absolute ethanol between    each set of surface wears to prevent carryover contamination. Allow    the alcohol to completely evaporate before proceeding. Replace the    foam liner and the cotton cloth, on the abrasion boat assembly,    between each set of surface wears.-   5. Alternate dry-wears and wet-wears. For wet-wears, spray clean    cotton cloth with sterile distilled water, using a Preval sprayer,    from a distance of 75±1 cm for not more than one second. Immediately    attach the moistened abrasion boat assembly to the abrasion tester,    and perform one cycle of surface wears.-   6. Wait at least 15 minutes after each wear to reinoculate surfaces.    Reinoculate test and control surfaces by applying 10 μL of the    Reinoculation Suspension (Step 3 in Test System Preparation) and    spread the inoculum to within ⅛ inch of the surface edge with a the    pipette tip. Dry at least 30 minutes at room temperature before    proceeding with the next wear.-   7. The period between test substance application and the initiation    of the sanitizer test (carriers into neutralizer broth) must be at    least 24 hours.

Enumeration of Survivors

-   1. With the Final Inoculum Suspension (Step 4 in Test System    Preparation), inoculate the first surface with 10 μL, at time zero.    Begin inoculation about 5 seconds before time zero, and spread    aliquot over the surface so it is completed at time zero. Begin the    inoculation of the second surface similarly, at given intervals,    until all test and control surfaces have been inoculated.-   2. At 5 minutes (or other appropriate time) use alcohol-flamed    forceps to transfer the surfaces to 30 mL of neutralizer broth in a    50 mL centrifuge tube. Repeat until all test and control surfaces    have been completed.-   3. Sonicate the samples for 20±2 seconds in a sonicating water bath.    Then agitate the samples on an orbital shaker for 3-4 minutes at 250    rpm.-   4. Serially dilute the control sample suspensions in PBDW and    prepare duplicate pour plates of the 10⁻², 10⁻³ and 10⁻⁴ dilutions.    The control plates must have a minimum of 1×10⁴ CFU/mL for a valid    test.-   5. Serially dilute the test sample suspensions in PBDW and prepare    duplicate pour plates of the 10⁰, 10⁻² and 10⁻⁴ dilutions.-   6. Plate all samples within 30 minutes of their transfer to    neutralizer broth.

Method Parameters: Test Substance Formula:

Test composition C Raw Material Concentration Bardac DM50 (Alkyl benzylquat 50% active)) 50% Vantocil P (PHMG 20% active) 50%

Test Substance Dilutions:

Prepared on Jan. 20, 2009: Desired Test Solution (Volume of TestConcentration Diluent Substance/Total Volume) 1:64 410 ppm Synthetic1.56 mL/100 mL Hard Water (pH 7.65) 1:64 510 ppm Synthetic 1.56 mL/100mL Hard Water (pH 7.84)

-   -   Test Systems: Staphylococcus aureus ATCC 6538        -   Klebsiella pneumoniae ATCC 4352        -   Staphylococcus aureus (MRSA) ATCC 33592        -   Enterococcus faecalis (VRE) ATCC 51299    -   Soil Challenge: 5% Bovine Serum    -   Test Material: 1″×1″ Glass Surfaces        -   1 mm thick non-frosted microscope slides, cut into squares    -   Test Substance Application: 50 μL applied and spread end to end        with a pipette tip    -   Number of Wears Performed: 12 Total (6 dry and 6 wet)    -   Number of Re-inoculations Performed: 5 Total    -   Neutralizer Medium: 30 mL Dey Engley Broth        -   A neutralizer screen was performed as part of the testing,            verified that the neutralizer adequately neutralized the            product and was not detrimental to the tested organisms.    -   Plating Medium: Tryptone Glucose Extract Agar    -   Incubation: 35° C. for 48 hours

Inoculum Numbers Inoculum Average Test System Suspension A B CFU/mL MRSAInitial 148 × 10³  155 × 10³ 1.5 × 10⁵ ATCC 33592 Reinoculation 61 × 10³ 45 × 10³ 5.3 × 10⁴ Final 124 × 10⁶  134 × 10⁶ 1.3 × 10⁸ VRE Initial 34× 10⁴  35 × 10⁴ 3.5 × 10⁵ ATCC 51299 Reinoculation 94 × 10³ 136 × 10³1.2 × 10⁵ Final 50 × 10⁷  35 × 10⁷ 4.2 × 10⁸

MRSA ATCC 33592 Test Survivors Survivors Average Percent Substance(CFU/mL) (CFU/Carrier)* CFU/Carrier Reduction TritonX 253 × 10², 263 ×10² 7.59 × 10⁵, 7.89 × 10⁵ 7.8 × 10⁵ N/A 268 × 10², 251 × 10² 8.04 ×10⁵, 7.53 × 10⁵ Test 0 × 10⁰, 0 × 10⁰ <30, <30, <30, <30 >99.996Composition C 0 × 10⁰, 1 × 10⁰ 3.00 × 10¹ *CFU/mL × 30

VRE ATCC 51299 Test Survivors Survivors Average Percent Substance(CFU/mL) (CFU/Carrier)* CFU/Carrier Reduction TritonX 67 × 10³, 90 × 10³2.01 × 10⁶, 2.70 × 10⁶  2.2 × 10⁶ N/A 67 × 10³, 66 × 10³ 2.01 × 10⁶,1.98 × 10⁶ Test 4 × 10², 4 × 10² 1.20 × 10⁴, 1.20 × 10⁴ <6.0 ×10³ >99.727 Composition C 0 × 10⁰, 0 × 10⁰ <30, <30 *CFU/mL × 30

-   Test Date: 01-22-2009-   Date Results Read: 01-26-2009

Inoculum Numbers Inoculum Average Test System Suspension A B CFU/mLStaphylococcus Initial 114 × 10³ 100 × 10³ 1.1 × 10⁵ aureusReinoculation  43 × 10³  55 × 10³ 4.9 × 10⁴ ATCC 6538 Final 111 × 10⁶112 × 10⁶ 1.1 × 10⁸ Klebsiella Initial 117 × 10³ 142 × 10³ 1.3 × 10⁵pneumonias Reinoculation  55 × 10³  60 × 10³ 5.8 × 10⁴ ATCC 4352 Final109 × 10⁷ 129 × 10⁷ 1.2 × 10⁹

Staphylococcus aureus ATCC 6538 Test Survivors Survivors Average PercentSubstance (CFU/mL) (CFU/Carrier)* CFU/Carrier Reduction TritonX 30 ×10³, 19 × 10³ 9.00 × 10⁵, 5.70 × 10⁵ 8.0 × 10⁵ N/A 31 × 10³, 26 × 10³9.30 × 10⁵, 7.80 × 10⁵ Test 0 × 10⁰, 0 × 10⁰ <30, <30, <30, <30<30 >99.996 Composition C 0 × 10⁰, 0 × 10⁰ *CFU/mL × 30

Klebsiella pneumoniae ATCC 4352 Test Survivors Survivors Average PercentSubstance (CFU/mL) (CFU/Carrier)* CFU/Carrier Reduction TritonX 22 ×10³, 18 × 10³ 6.60 × 10⁵, 5.40 × 10⁵ 3.9 × 10⁵ N/A 48 × 10², 75 × 10²1.44 × 10⁵, 2.25 × 10⁵ Test 0 × 10⁰, 0 × 10⁰ <30, <30, <30, <30<30 >99.992 Composition C 0 × 10⁰, 0 × 10⁰ *CFU/mL × 30

Conclusion:

To be defined as a sanitizer, the test substances on the hard inanimatesurface must reduce the total number of organisms by at least 99.9% onthe surface within a 5 minute period (after the final inoculation).

Test Composition C passed with a greater than 99.9% reduction in S.aureus, K. pneumoniae and MRSA numbers, and almost passed against VREwith a greater than 99.7% reduction.

Example 4 Residual Hard Surface Virucidal Efficacy of Test Composition C

The objective of this analysis was to examine the virucidal efficacy ofBiocide X, a residual product according to the invention), againstHerpes Simplex I virus after application to inanimate, non-porous,non-food contact surfaces. A test method was developed based on theResidual Self-Sanitizing Activity on Hard, Non-Porous Surfaces method,but adapted for viruses.

Test Method: Residual Self-Virucidal Activity on Hard, Non-PorousSurfaces

-   -   Test Surface Preparation:    -   Clean glass test surfaces were sterilized in an autoclave. The        test surfaces were placed in sterile glass petri dishes        containing 2 layers of Whatman No. 2 paper. Each petri dish had        two coupons in them.    -   Test Surface Inoculation and Coating:    -   10 μL of thawed viral stock was spread evenly over the entire        coupon. The coupons were dried at ambient temperature for 30        minutes with the lids cracked open. 50 μL of the test substance        was spread evenly onto the coupons, and 50 μL of 0.0 I % Triton        X 100 was gently spread over the surface of the control coupons        (2 coupons per treatment). The coupons were dried overnight at        ambient temperature in a biological safety cabinet with the lids        closed.    -   Operating Technique:        -   12 wears were performed using the Gardner Abrasion Tester.            The wears alternated between dry or wet, beginning with a            dry wear and ending with a wet wear. The abrasion boat was            assembled in the following manner for each wear: a thin foam            pad and a cotton strip were wrapped around the flat surface            of the boat. For dry wears, the boat was run over two            coupons for one cycle. For wet wears, the boat was sprayed            for 1 second with sterile water (in a Prevail sprayer) at a            distance of about 75 cm before it was run over two coupons            for one cycle. The coupons were reinoculated with 10 μL of            thawed viral stock (stock was refrigerated when not in use),            spread evenly and dried at ambient temperature until visibly            dry. Coupons were reinoculated after the first 5 wears.            Approximately 5 minutes elapsed after the previous wear            before the coupons were reinoculated. After the final wear,            10 μL of a new viral stock was spread over the surface of            each coupon. After 9 minutes of exposure, 200 μL of cell            culture medium was place on top of each coupon and scraped            with a cell scraper. At the end of the exposure time of 10            minutes, 100 μL of the scraped cells/cell culture medium            were added to a prepared GE column and centrifuged for I            minute, and then added to 900 μL of Fetal Bovine Serum (10.2            dilution). Each coupon was serially diluted in cell culture            medium and added to prepared 24 well cell culture plates            seeded with the appropriate cell line for the virus tested.            The plates were examined for Cytopathic Effects (CPE) after            7 days of incubation at 35±2° C. with 5% CO₂.

Method Parameters:

Test Substance: Test Composition C Virus: Herpes Simplex 1 Cell Line:Vero Cells Number of Wears: 12 Test Surface: 1 inch × 1 inch squareglass slides Organic Soil: 5% Fetal Bovine Serum (FBS) Final InoculumExposure Time: 10 minutes Exposure Temperatures: Ambient Neutralizer: GESephacryl & Fetal Bovine Serum Cell Culture Media: Eagle's MinimumEssential Medium (EMEM) with 5% Fetal Bovine Serum Incubation: 7 days at35 ± 2° C., 5% CO₂

Results:

Virus Virus Test Test Control Control Composition C Composition CDilution Rep 1* Rep 2* Residual Rep 1 Residual Rep 2 Cell Control 0 0 00 0 0 0 0 0 0 0 0 0 0 0 0 10⁻² + + + + + + + + 0 0 0 0 0 0 0 010⁻³ + + + + + + + + 0 0 0 0 0 0 0 0 10⁻⁴ 0 + + 0 + + + + 0 0 0 0 0 0 00 10⁻⁵ 0 0 0 0 + + 0 0 0 0 0 0 0 0 0 0 10⁻⁶ 0 0 0 0 0 0 0 0 0 0 0 0 0 00 0 TCID₅₀/0.1 mL 10^(4.0) 10^(5.0) ≦10^(1.5) ≦10^(1.5) Log₁₀ Reduction ≧3.0  ≧3.0 *Results averaged (10^(4.5)) and used to calculate the Log₁₀Reduction

Test Composition C Test Composition C Dilution Neutralizer CytotoxicityCell Control 0 0 0 0 10⁻² + + 0 0 10⁻³ + + 0 0 10⁻⁴ + 0 0 0 10⁻⁵ + + 0 010⁻⁶ 0 0 0 0 + = Positive for the presence of the test virus 0 =Negative for the presence of the virus and/or no cytotoxicity present

Conclusion:

The average of the viral titer in this test was 10^(4.5), and TestComposition C Residual showed complete inactivation of the HerpesSimplex 1 virus for a log reduction of ≧3.0. While there are no specificstandards for effective Residual Self-Virucidal products/claims, thesetest results would meet the current hard surface virucidal standards.

Example 5 100-Wear Residual Hard Surface Screen of Test Composition CObjective:

The objective of the analysis was to examine the residual hard surfaceefficacy of Test Composition C against Staphylococcus aureus ATCC 6538and Klebsiella pneumoniae ATCC 4352 after application to inanimate,non-porous, non-food contact surfaces.

Test Method: Residual Self-Sanitizing Activity on Hard, Non-PorousSurfaces Test System Preparation

-   1. At least three consecutive loop transfers of a 24 hour culture of    Staphylococcus aureus ATCC 6538 and Klebsiella pneumoniae ATCC 4352    were performed in 10 mL of AOAC Synthetic broth or AOAC Nutrient    broth (respectively) and incubated at 35° C.-   2. Final Inoculum Suspension:    -   2.1. Vortex an 18-24 hour culture for 3-4 seconds.    -   2.2. Make one 1/10 dilution in sterile PBDW and vortex.    -   2.3. Add organic soil load to equal 5%, vortex, and let stand        for 15±1 minutes.

Test Surface Preparation

-   1. Clean glass surfaces by rinsing in alcohol, then sterile water,    and allow to air dry.-   2. Decontaminate glass surfaces by immersing in absolute ethanol and    allowing to air dry (slides can be autoclaved if necessary).-   3. Transfer to individual glass petri dishes lined with 1-2 layers    of sterile Whatman No. 2 paper, and allow all surfaces to dry    completely prior to use (approximately one day).-   4. Apply the test substance to the test surfaces on a clean dry    surface. Apply test substance to each test surface appropriate to    the application instructions. If no application method is specified,    apply 50 μL of the test substance to the test surface and spread,    with a sterile disposable loop, in an even layer over entire test    surface. Allow the surfaces to dry overnight, covered, at room    temperature.-   5. Apply a 0.01% TritonX 100 solution (made and filter sterilized on    the day of application) to each of the control surfaces in the same    manner as the test substances. Allow the control surfaces to dry    under the same conditions as the test surfaces.

Operating Technique

-   1. Wear and Reinoculation of Test and Control Surfaces: The treated    surfaces will undergo a wear and reinoculation regimen, which will    take place over at least a 24 hour period at room temperature.-   2. GardCo Washability and Wear Tester: A cycle equals one pass to    the left, and a return pass to the right. One pass on the abrasion    tester should provide a contact time with the surfaces of    approximately 2 seconds.-   3. Place one set of surfaces (two test or control surfaces) into the    cut out region of the surface wear area on the abrasion tester, and    perform 100 cycles of surface wears.-   4. Decontaminate the surface wear area with absolute ethanol between    each set of surface wears to prevent carryover contamination. Allow    the alcohol to completely evaporate before proceeding. Replace the    foam liner and the cotton cloth, on the abrasion boat assembly,    between each set of surface wears.-   5. The period between test substance application and the initiation    of the sanitizer test (carriers into neutralizer broth) must be at    least 24 hours.

Enumeration of Survivors

-   1. With the Final Inoculum Suspension (Step 2 in Test System    Preparation), inoculate the first surface with 10 μL, at time zero.    Begin inoculation about 5 seconds before time zero, and spread    aliquot over the surface so it is completed at time zero. Begin the    inoculation of the second surface similarly, at given intervals,    until all test and control surfaces have been inoculated.-   2. At 5 minutes (or other appropriate time) use alcohol-flamed    forceps to transfer the surfaces to 30 mL of neutralizer broth in a    50 mL centrifuge tube. Repeat until all test and control surfaces    have been completed.-   3. Sonicate the samples for 20±2 seconds in a sonicating water bath.    Then agitate the samples on an orbital shaker for 3-4 minutes at 250    rpm.-   4. Serially dilute the control sample suspensions in PBDW and    prepare duplicate pour plates of the 10⁻², 10⁻³ and 10⁻⁴ dilutions.    The control plates must have a minimum of 1×10⁴ CFU/mL for a valid    test.-   5. Serially dilute the test sample suspensions in PBDW and prepare    duplicate pour plates of the 10⁰, 10⁻² and 10-4 dilutions.-   6. Plate all samples within 30 minutes of their transfer to    neutralizer broth.

Method Parameters: Test Substance Formula:

Test composition C Raw Material Concentration Bardac DM50 (Alkyl benzylquat 50% active)) 50% Vantocil P (PHMG 20% active) 50%

-   -   Test Systems: Staphylococcus aureus ATCC 6538        -   Klebsiella pneumoniae ATCC 4352    -   Soil Challenge: 5% Bovine Serum    -   Test Material: 1″×1″ Glass Surfaces        -   1 mm thick non-frosted microscope slides, cut into squares    -   Test Substance Application: 50 μL applied and spread end to end        with a pipette tip—wiped on with a soft cloth    -   Number of Wears Performed: 100 per slide set    -   Number of Re-inoculations Performed: None    -   Neutralizer Medium: 20 mL Dey Engley Broth        -   A neutralizer screen was performed as part of the testing,            verified that the neutralizer adequately neutralized the            product and was not detrimental to the tested organisms.    -   Plating Medium: Tryptone Glucose Extract Agar    -   Incubation: 35° C. for 48 hours

Inoculum Numbers Inoculum Average Test System Suspension A B CFU/mLStaphylococcus Final  83 × 10⁶ 84 × 10⁶ 8.4 × 10⁷ aureus ATCC 6538Klebsiella Final 102 × 10⁶ 86 × 10⁶ 9.4 × 10⁷ pneumoniae ATCC 4352

Staphylococcus aureus ATCC 6538 Average Log Geometric Percent TestSubstance Rep CFU/mL CFU/Carrier* Growth Mean Reduction Triton X 1  36 ×10³ 7.20 × 10⁵ 5.76 5.8 × 10⁵ N/A  21 × 10³ 4.20 × 10⁵ Test 1 138 × 10²2.76 × 10⁵ 5.42 3.6 × 10⁵ 37.931 composition 123 × 10² 2.46 × 10⁵ C(pipette 2 251 × 10² 5.02 × 10⁵ 5.69 application) 243 × 10² 4.86 × 10⁵Test 1 362 × 10² 7.24 × 10⁵ 5.82 7.1 × 10⁵ 0.000 composition 293 × 10²5.86 × 10⁵ C (wipe 2 367 × 10² 7.34 × 10⁵ 5.88 application) 386 × 10²7.72 × 10⁵ *Average CFU/mL × Total Volume of Neutralized Test Substance(20 mL).

Klebsiella pneumoniae ATCC 4352 Average Log Geometric Percent TestSubstance Rep CFU/mL CFU/Carrier* Growth Mean Reduction Triton X 1  37 ×10³ 7.40 × 10⁵ 5.84 6.9 × 10⁵ N/A  32 × 10³ 6.40 × 10⁵ Test 1 189 × 10²3.78 × 10⁵ 5.60 3.8 × 10⁵ 44.928 composition 205 × 10² 4.10 × 10⁵ C(pipette 2 186 × 10² 3.72 × 10⁵ 5.55 application) 171 × 10² 3.42 × 10⁵Test 1 235 × 10² 4.70 × 10⁵ 5.67 4.5 × 10⁵ 34.783 composition 238 × 10²4.76 × 10⁵ C (wipe 2 215 × 10² 4.30 × 10⁵ 5.63 application) 214 × 10²4.28 × 10⁵ *Average CFU/mL × Total Volume of Neutralized Test Substance(20 mL).

Conclusion:

To be defined as a sanitizer, the test substance on the hard inanimatesurface must reduce the total number of organisms by at least 99.9%(based on the Geometric Mean) on the surface with a 5 minute period(after the final inoculation).

None of the Test composition C samples (pipette application and wipeapplication passed the 100 wear screen, against S. aureus or K.pneumoniae, after a 5 minute final exposure. Don't know if it iscritical to include this slide other than to represent the transientnature of the antimicrobial treatment on the surface.

Example 6 Residual Self-Sanitizing Screen of Test Composition C in aConcentration Gradient Objective:

The objective of the analysis was to examine the residualself-sanitizing efficacy of Test composition C in a concentrationgradient against Staphylococcus aureus ATCC 6538 and Klebsiellapneumonia ATCC 4352 after application to inanimate, non-porous, non-foodcontact surfaces.

Test Method: Residual Self-Sanitizing Activity on Hard, Non-PorousSurfaces Test System Preparation

-   1. At least three consecutive loop transfers of a 24 hour culture of    Staphylococcus aureus ATCC 6538 and Klebsiella pneumoniae ATCC 4352    were performed in 10 mL of AOAC Synthetic broth of AOAC Nutrient    broth (respectively) and incubated at 35° C.-   2. Initial Inoculum Suspension:    -   2.1 Vortex a 48-54 hour culture for 3-4 seconds.    -   2.2 Make two 1/100 dilutions in sterile PBDW and let stand for        15±1 minutes.-   3. Final Inoculum Suspension:    -   3.1 Vortex an 18-24 hour culture for 3-4 seconds.    -   3.2 Make one 1/10 dilution in sterile PBDW and vortex.    -   3.3 Add organic soil load to equal 5%, vortex, and let stand for        15±1 minutes.

Test Surface Preparation

-   1. Test surfaces were cleaned and autoclaved glass coverslips.-   2. Inoculation of Test Surfaces    -   2.1 Apply a 10 μL aliquot of the Initial Inoculum Suspension        (Step 2 in the Test System Preparation) to each of 2-4 prepared        test surfaces, per test substance, per test system. Also        inoculate 2-4 prepared test surfaces, per test system, to be        used as control surfaces.    -   2.2 Spread inoculum to within ⅛ inch of the edge with the        pipette tip.    -   2.3 Dry with lids cracked at 35° C. for 30-35 minutes, or until        visibly dry.    -   2.4 Apply the test substance to the test surfaces on a clean dry        surface. Apply test substance to each test surface appropriate        to the application instructions. If no application method is        specified, apply 50 μL of the test substance to the test surface        and spread, with a sterile disposable lop, in an even layer over        entire test surface. Allow the surface to dry overnight,        covered, at room temperature.-   3. Apply a 0.01% TritonX 100 solution (made and filter sterilized on    the day of application) to each of the control surfaces in the same    manner as the test substances. Allow the control surfaces to dry    under the same conditions as the test surfaces.

Operating Technique

-   1. The period between test substance application and the initiation    of the sanitizer test (carriers into neutralizer broth) must be at    least 24 hours.-   2. With the Final Inoculum Suspension (Step 4 in Test System    Preparation), inoculate the first surface with 10 μL, at time zero.    Begin inoculation about 5 seconds before time zero, and spread    aliquot over the surface so it is completed at time zero. Begin the    inoculation of the second surface similarly, at given intervals,    until all test and control surfaces have been inoculated.-   3. At 5 minutes (or other appropriate time) use alcohol-flamed    forceps to transfer the surfaces to 30 mL of neutralizer broth in a    50 mL centrifuge tube. Repeat until all test and control surfaces    have been completed.-   4. Sonicate the samples for 20±2 seconds in a sonicating water bath.    Then agitate the samples on an orbital shaker for 3-4 minutes at 250    rpm.

Enumeration of Survivors

-   1. Serially dilute the control sample suspensions in PBDW and    prepare duplicate pour plates of the 10⁻², 10⁻³ and 10⁻⁴ dilutions.    The control plates must have a minimum of 1×10⁴ CFU/mL for a valid    test.-   2. Serially dilute the test sample suspensions in PBDW and prepare    duplicate pour plates of the 10⁰, 10⁻² and 10⁻⁴ dilutions.-   3. Plate all samples within 30 minutes of their transfer to    neutralizer broth.

Method Parameters: Test Substance Formula:

Raw Materials Concentration Test composition C - Phase Stable Vantocil P(HMBG)  50% Barquat DM50  50% Commercial Quaternary Disinfectant Bardac208M 12.5% DI Water 87.5%

Test Substance Dilutions:

Test Solution Test Desired (Volume of Test Substance ConcentrationDiluent Substance/Total Volume) Test 1:64  420 ppm 1.56 mL/100 mLcomposition 1:96  Synthetic 1.04 mL/100 mL C 1:128 Hard Water 0.78mL/100 mL 1:256 (pH 7.98) 0.78 mL/200 mL Commercial 1:256 0.78 mL/200 mLQuaternary 1:512 0.39 mL/200 mL Disinfectant

-   -   Test Systems: Staphylococcus aureus ATCC 6538        -   Klebsiella pneumoniae ATCC 4352    -   Soil Challenge: 5% Bovine Serum    -   Test Material: 1″×1″ Glass Coverslip    -   Test Substance Application: 50 μL spread with a pipette tip    -   Neutralizer Medium: 30 mL Dey Engley Broth        -   A neutralizer screen was performed as part of the testing,            verified that the neutralizer adequately neutralized the            product and was not detrimental to the tested organisms.    -   Plating Medium: Tryptone Glucose Extract Agar    -   Incubation: 35° C. for 48 hours

Inoculum Numbers Inoculum Average Test System Suspension A B CFU/mLStaphylococcus Initial 112 × 10³ 145 × 10³ 1.3 × 10⁵ aureus Final  80 ×10⁶  92 × 10⁶ 8.6 × 10⁷ ATCC 6538 Klebsiella Initial  77 × 10³  87 × 10³8.2 × 10⁴ pneumoniae Final 116 × 10⁶ 105 × 10⁶ 1.1 × 10⁸ ATCC 4352

Staphylococcus aureus ATCC 6538 Test Survivors Survivors Average PercentSubstance (CFU/mL) (CFU/Carrier)* CFU/Carrier Reduction TritonX 158 ×10², 178 × 10² 4.74 × 10⁵, 5.34 × 10⁵ 7.9 × 10⁵ N/A 33 × 10³, 39 × 10³9.90 × 10⁵, 1.17 × 10⁶ Test 0 × 10⁰, 0 × 10⁰ <30, <30, <30, <30<30 >99.996 Composition C 0 × 10⁰, 0 × 10⁰ (1:64) Test 3 × 10², 4 × 10²9.00 × 10³, 1.20 × 10⁴ 2.0 × 10⁴ 97.468 Composition C 10 × 10², 10 × 10²3.00 × 10⁴, 3.00 × 10⁴ (1:94) Test 162 × 10⁰, 158 × 10⁰ 4.86 × 10³, 4.74× 10³ 6.3 × 10³ 99.202 Composition C 271 × 10⁰, 255 × 10⁰ 8.13 × 10³,7.65 × 10³ (1:128) Test 101 × 10⁰, 127 × 10⁰ 3.03 × 10³, 3.81 × 10³ 3.8× 10³ 99.519 composition C 137 × 10⁰, 142 × 10⁰ 4.11 × 10³, 4.26 × 10³(1:256) *CFU/mL × 30

ebsiella pneumoniae ATCC 4352 Test Survivors Survivors Average PercentSubstance (CFU/mL) (CFU/Carrier)* CFU/Carrier Reduction TritonX 176 ×10², 189 × 10² 5.28 × 10⁵, 5.67 × 10⁵  5.0 × 10⁵ N/A 145 × 10², 159 ×10² 4.35 × 10⁵, 4.77 × 10⁵ Test 0 × 10⁰, 0 × 10⁰ <30, <30, <30, <30 <1.3× 10² >99.974 Composition C 5 × 10⁰, 0 × 10⁰ 1.50 × 10², 3.00 × 10²(1:64) Test 2 × 10², 3 × 10² 6.00 × 10³, 9.00 × 10³  4.6 × 10³ 99.080Composition C 57 × 10⁰, 53 × 10⁰ 1.71 × 10³, 1.59 × 10³ (1:94) Test 0 ×10⁰, 0 × 10⁰ <30, <30 <1.1 × 10² >99.987 Composition C 3 × 10⁰, 7 × 10⁰9.00 × 10¹, 2.80 × 10² (1:128) Test 41 × 10⁰, 35 × 10⁰ 1.23 × 10³, 1.05× 10³ <5.8 × 10² >99.884 composition C 0 × 10⁰, 0 × 10⁰ <30, <30 (1:256)*CFU/mL × 30

Conclusion:

To be defined as a sanitizer, the test substances on the hard inanimatesurface must reduce the total number of organisms by at least 99.9%(based on the Geometric Mean) on the surface within a 5 minute period(after the final inoculation).

Against S. auerus, only the 1:64 dilution of test composition C X passedwith a greater than 99.9 percent reduction.

Against K. Pneumoniae, the 1:64 and 1:128 dilutions of test compositionC passed with a greater than 99.9 percent reduction.

Example 7 Residual Hard Surface Test Against PHMB:QAC Objective:

The objective of the analysis was to examine the residual hard surfaceefficacy of PHMB:QAC against Staphylococcus aureus ATCC 6538 andKlebsiella pneumoniae ATCC 4352 after application to inanimate,non-porous, non-food contact surfaces.

Test Method: Residual Self-Sanitizing Activity on Hard, Non-PorousSurfaces Test System Preparation

-   1. At least three consecutive loop transfers of a 24 hour culture of    Staphylococcus aureus ATCC 6538 and Klebsiella pneumoniae ATCC 4352    were performed in 10 mL of AOAC Synthetic broth or AOAC Nutrient    broth (respectively) and incubated at 35° C.-   2. Final Inoculum Suspension:    -   2.1. Vortex an 18-24 hour culture for 3-4 seconds.    -   2.2. Add organic soil load to equal 5%, vortex, and let stand        for 15±1 minutes.

Test Surface Preparation

-   1. Clean glass surfaces by rinsing in alcohol, then sterile water,    and allow to air dry.-   2. Decontaminate glass surfaces by immersing in absolute ethanol and    allowing to air dry (slides can be autoclaved if necessary).-   3. Transfer to individual glass petri dishes lined with 1-2 layers    of sterile Whatman No. 2 paper, and allow all surfaces to dry    completely prior to use (approximately one day).-   4. Apply the test substance to the test surfaces on a clean dry    surface. Apply test substance to each test surface appropriate to    the application instructions. If no application method is specified,    apply 50 μL of the test substance to the test surface and spread,    with a sterile disposable loop, in an even layer over entire test    surface. Allow the surfaces to dry overnight, covered, at room    temperature.-   5. Apply a 0.01% TritonX 100 solution (made and filter sterilized on    the day of application) to each of the control surfaces in the same    manner as the test substances. Allow the control surfaces to dry    under the same conditions as the test surfaces.

Operating Technique

-   1. Wear and Reinoculation of Test and Control Surfaces: The treated    surfaces will undergo a wear and reinoculation regimen, which will    take place over at least a 24 hour period at room temperature.-   2. GardCo Washability and Wear Tester: A cycle equals one pass to    the left, and a return pass to the right. One pass on the abrasion    tester should provide a contact time with the surfaces of    approximately 2 seconds.-   3. Place one set of surfaces (two test or control surfaces) into the    cut out region of the surface wear area on the abrasion tester, and    perform 100 cycles of surface wears.-   4. Decontaminate the surface wear area with absolute ethanol between    each set of surface wears to prevent carryover contamination. Allow    the alcohol to completely evaporate before proceeding. Replace the    foam liner and the cotton cloth, on the abrasion boat assembly,    between each set of surface wears.-   5. The period between test substance application and the initiation    of the sanitizer test (carriers into neutralizer broth) must be at    least 24 hours.

Enumeration of Survivors

-   1. With the Final Inoculum Suspension (Step 2 in Test System    Preparation), inoculate the first surface with 10 μL, at time zero.    Spot inoculate over the entire surface. Begin the inoculation of the    second surface similarly, at given intervals, until all test and    control surfaces have been inoculated.-   2. At 5 minutes (or other appropriate time) use alcohol-flamed    forceps to transfer the surfaces to 30 mL of neutralizer broth in a    50 mL centrifuge tube. Repeat until all test and control surfaces    have been completed.-   3. Vortex the samples on high for one minute.-   4. Serially dilute the control sample suspensions in PBDW and    prepare duplicate pour plates of the 10⁻², 10⁻³ and 10⁻⁴ dilutions.    The control plates must have a minimum of 1×10⁴ CFU/mL for a valid    test.-   5. Serially dilute the test sample suspensions in PBDW and prepare    duplicate pour plates of the 10⁰, 10⁻² and 10-4 dilutions.-   6. Plate all samples within 30 minutes of their transfer to    neutralizer broth.

Method Parameters:

Test Substance Formulas:

-   -   1:1 PHMB:QAC    -   1:4 PHMB:QAC    -   4:1 PHMB:QAC

Ratios based on raw material percentages (e.g. PHMB is 20% and QAC is50% active) Adjusting for activity testing compositions were

-   -   1:2.5 PHMB:QAC (most preferred)    -   1:10 PHMB:QAC (more preferred)    -   1:0.625 PHMB:QAC (preferred)    -   Test Systems: Staphylococcus aureus ATCC 6538        -   Klebsiella pneumoniae ATCC 4352    -   Soil Challenge: 5% Bovine Serum    -   Test Material: 1″×1″ Glass Surfaces        -   1 mm thick non-frosted microscope slides, cut into squares    -   Test Substance Application: 50 μL spread end to end with a        pipette tip    -   Number of Wears Performed: 12, 24 and 48    -   Number of Re-inoculations: None    -   Neutralizer Medium: 30 mL Dey Engley Broth        -   A neutralizer screen was performed as part of the testing,            verified that the neutralizer adequately neutralized the            product and was not detrimental to the tested organisms.    -   Plating Medium: Tryptone Glucose Extract Agar    -   Incubation: 35° C. for 48 hours

Inoculum Numbers Inoculum Average Test System Suspension A B CFU/mLStaphylococcus Final 116 × 10⁷ 67 × 10⁷ 9.2 × 10⁸ aureus ATCC 6538Klebsiella Final  66 × 10⁷ 87 × 10⁷ 7.6 × 10⁸ pneumoniae ATCC 4352

Staphylococcus aureus ATCC 6538 Average # Log Geometric Percent TestSubstance Wears CFU/mL CFU/Carrier* Growth Mean Reduction Triton X NA    151 × 10³    30 × 10⁶  6.64 47 × 10⁶ NA     191 × 10³   5.7 × 10⁶ NA     167 × 10³   5.0 × 10⁶  6.70     171 × 10³   5.1 × 10⁶  1:1 12  0, 0 × 10⁰ <3.0 × 10¹  1.48 3.0 × 10¹ 99.999 PHMB:QAC   0, 0 × 10⁰<3.0 × 10¹  1.48 24   4, 7 × 10⁰   1.6 × 10²  2.20 6.9 × 10¹ 99.998   0,0 × 10⁰ <3.0 × 10¹  1.48 48 258, 262 × 10²   7.8 × 10⁵  5.89 8.2 × 10⁵82.553 308, 268 × 10²   8.7 × 10⁵  5.94 1:4 12   0, 0 × 10⁰ <3.0 × 10¹ 1.48 2.0 × 10² 99.996 PHMB:QAC  12, 14 × 10⁰   1.3 × 10³  3.11 24   0, 0× 10⁰ <3.0 × 10¹  1.48 5.0 × 10² 99.989  93, 70 × 10²   8.2 × 10³  3.9148 430, 442 × 10²   1.3 × 10⁶  6.11 1.5 × 10⁶ 68.085 508, 620 × 10²  1.7 × 10⁶  6.23 4:1 12   7, 7 × 10⁰   2.1 × 102  2.32 7.9 × 10¹ 99.998PHMB:QAC   0, 0 × 10⁰ <3.0 × 101  1.48 24 596, 572 × 10⁰   1.7 × 10⁴ 4.23 1.5 × 10⁴ 99.681 504, 420 × 10⁰   1.4 × 10⁴  4.15 48  53, 52 × 10²  1.6 × 10⁵  5.20 9.2 × 10⁴ 98.042  16, 20 × 10²   5.4 × 10⁴  4.73*Average CFU/mL × Total Volume of Neutralized Test Substance (30 L).

Conclusion:

To be defined as a sanitizer, the test substances on the hard inanimatesurface must reduce the total number of organisms by at least 99.9%(based on the Geometric Mean) on the surface within a 5 minute period(after the final inoculation).

The 4:1 ratio of PHMB:QAC had the best durability over time with 98%reduction of 48 wears.

Example 8 Residual Hard Surface Test Against PHMB and QAC AloneObjective:

The objective of the analysis was to examine the residual hard surfaceefficacy of QAC, PHMB and a conventional commercial disinfectant(Disinfectant B) vs. the Test Composition A (PHMB:QAC blend) againstStaphylococcus aureus ATCC 6538 after application to inanimate,non-porous, non-food contact surfaces.

Test Method: Residual Self-Sanitizing Activity on Hard, Non-PorousSurfaces Test System Preparation

-   1. At least three consecutive loop transfers of a 24 hour culture of    Staphylococcus aureus ATCC 6538 and Klebsiella pneumoniae ATCC 4352    were performed in 10 mL of AOAC Synthetic broth or AOAC Nutrient    broth (respectively) and incubated at 35° C.-   2. Final Inoculum Suspension:    -   2.1. Vortex an 18-24 hour culture for 3-4 seconds.    -   2.2. Make one 1/10 dilution in sterile PBDW and vortex.    -   2.3. Add organic soil load to equal 5% vortex, and let stand for        15±1 minutes.

Test Surface Preparation

-   1. Clean glass surfaces by rinsing in alcohol, then sterile water,    and allow to air dry.-   2. Decontaminate glass surfaces by immersing in absolute ethanol.    Transfer to individual glass petri dishes lined with 1-2 layers of    sterile Whatman No. 2 paper, and allow all surfaces to dry    completely prior to use (approximately one day). Slides can be    autoclaved if desired.-   3. Apply the test substance to the test surfaces on a clean dry    surface. Apply test substance to each test surface appropriate to    the application instructions. If no application method is specified,    apply 50 μL of the test substance to the test surface and spread,    with a sterile disposable loop, in an even layer over entire test    surface. Allow the surfaces to dry overnight, covered, at room    temperature.-   5. Apply a 0.01% TritonX 100 solution (made and filter sterilized on    the day of application) to each of the control surfaces in the same    manner as the test substances. Allow the control surfaces to dry    under the same conditions as the test surfaces.

Operating Technique

-   1. Wear and Reinoculation of Test and Control Surfaces: The treated    surfaces will undergo a wear and reinoculation regimen, which will    take place over at least a 24 hour period at room temperature.-   2. GardCo Washability and Wear Tester: A cycle equals one pass to    the left, and a return pass to the right. One pass on the abrasion    tester should provide a contact time with the surfaces of    approximately 2 seconds.-   3. Place one set of surfaces (two test or control surfaces) into the    cut out region of the surface wear area on the abrasion tester, and    perform 100 cycles of surface wears.-   4. The period between test substance application and the initiation    of the sanitizer test (carriers into neutralizer broth) must be at    least 24 hours.

Enumeration of Survivors

-   1. With the Final Inoculum Suspension (Step 2 in Test System    Preparation), inoculate the first surface with 10 μL, at time zero.    Begin inoculation about 5 seconds before time zero. Spot the aliquot    over the surface so it is completed at time zero. Begin the    inoculation of the second surface similarly, at given intervals,    until all test and control surfaces have been inoculated.-   2. At 10 minutes use alcohol-flamed forceps to transfer the surfaces    to 20 mL of neutralizer broth in a sterile straight-sided jar.    Repeat until all test and control surfaces have been completed.-   3. Sonicate the samples for 20±2 seconds in a sonicating water bath.    Then agitate the samples on an orbital shaker for 4 minutes at 250    rpm.-   4. Serially dilute the control sample suspensions in PBDW and    prepare duplicate pour plates of the 10⁻², 10⁻³ and 10⁻⁴ dilutions.    The control plates must have a minimum of 1×10⁴ CFU/mL for a valid    test.-   5. Serially dilute the test sample suspensions in PBDW and prepare    duplicate pour plates of the 10⁰, 10⁻² and 10-4 dilutions.-   6. Plate all samples within 30 minutes of their transfer to    neutralizer broth.-   7. Incubate all plates and tubes at 35±2° C. for 48±4 hours.

Method Parameters:

-   -   Test Substances: Quaternary Disinfectant B—        -   Benzyl Quat (Bardac DM50)        -   PHMB (Vantocil P)

Test Substance Test concentration Diluent Disinfectant B Diluted to 3900ppm active QAC Sterile MilliQ 625 ppm active PHMB Water Bardac DM 50Diluted to 3900 ppm QAC Vantocil P Diluted to 625 ppm PHMB

-   -   Test Systems: Staphylococcus aureus ATCC 6538    -   Soil Challenge: 5% Bovine Serum    -   Test Material: 1″×1″ Glass Surfaces        -   1 mm thick non-frosted microscope slides, cut into squares    -   Test Substance Application: 50 μL applied and spread end to end        with a pipette tip    -   Number of Wears Performed: 10, 20, 30 and 40    -   Neutralizer Medium: 20 mL Dey Engley Broth        -   A neutralizer screen was performed as part of the testing,            verified that the neutralizer adequately neutralized the            product and was not detrimental to the tested organisms.    -   Plating Medium: Tryptone Glucose Extract Agar    -   Incubation: 35° C. for 48 hours

Results: Test Date: 05-12-2010 Date Results Read: 05-14-2010

Inoculum Numbers Test System A B Average CFU/mL Staphylococcus aureus115 × 10⁷ 111 × 10⁷ 1.13 × 10⁹ ATCC 6538

Staphylococcus aureus ATCC 6538 Test Log₁₀ Geometric Percent SubstanceWears Rep CFU/Carrier Growth Mean* Reduction Triton X 10 1   9.00 × 10⁶6.95 9.00 × 10⁶ N/A 2   7.00 × 10⁶ 6.84 20 1   5.00 × 10⁶ 6.70 9.00 ×10⁶ N/A 2   6.20 × 10⁶ 6.79 30 1   5.80 × 10⁶ 6.76 9.00 × 10⁶ N/A 2  1.60 × 10⁶ 6.20 40 1   2.00 × 10⁶ 6.30 9.00 × 10⁶ N/A 2   7.80 × 10⁶6.89 Quaternary 10 1   2.00 × 10¹ 1.30 9.00 × 10⁶ 99.952% Disinfectant 2  7.36 × 10⁵ 5.87 B 20 1   7.52 × 10⁵ 5.88 9.00 × 10⁶ 99.930% 2 <2.00 ×10¹ <1.30 30 1   1.60 × 10⁶ 6.20 9.00 × 10⁶ 95.087% 2   1.40 × 10⁴ 4.1540 1   2.00 × 10⁶ 6.30 9.00 × 10⁶ 57.634% 2   1.40 × 10⁶ 6.15 BardacDM50 10 1   1.60 × 10⁶ 6.20 9.00 × 10⁶ 91.689% 2   2.72 × 10⁵ 5.43 20 1  1.36 × 10⁵ 5.13 9.00 × 10⁶ 95.354% 2   4.92 × 10⁵ 5.69 30 1   1.96 ×10⁵ 5.29 9.00 × 10⁶ 99.935% 2 <2.00 × 10¹ <1.30 40 1   4.00 × 10³ 3.609.00 × 10⁶ 98.399% 2   1.00 × 10⁶ 6.00 Vantocil P 10 1   3.80 × 10⁶ 6.589.00 × 10⁶ 56.066% 2   3.20 × 10⁶ 6.50 20 1   2.20 × 10⁶ 6.34 9.00 × 10⁶68.479% 2   1.40 × 10⁶ 6.15 30 1   1.80 × 10⁶ 6.26 9.00 × 10⁶  5.541% 2  4.60 × 10⁶ 6.66 40 1   4.00 × 10⁶ 6.60 9.00 × 10⁶  0.000% 2   5.20 ×10⁶ 6.72 * = Antilog [(log₁ + log₂)/2]

Example 9

Bardac MB 50 is a commercially available alkyl dimethyl benzylquaternary ammonium chloride from Lonza Group Ltd,Muenchensteinerstrasse 38, CH-4002 Basel, Switzerland.

Bardac 205M is a commercially available blended linear alkyl quaternaryammonium chloride blend from Lonza Group Ltd, Muenchensteinerstrasse 38,CH-4002 Basel, Switzerland.

FMB AO-8 is a commercially available Octyl di-methyl amine oxide (40%active as concentrate) from Lonza Group Ltd, Muenchensteinerstrasse 38,CH-4002 Basel, Switzerland.

Versene 100 is a commercially available 40% solution of tetra sodiumethylene diamine tetra acetic acid from The Dow Chemical CompanyMidland, Mich. 48674 U.S.A.

Trilon M is a commercially available trisodium salt ofmethylglycinediacetic acid (Na3MGDA) available from BASF Corporation,100 Campus Drive, Florham Park, N.J. 07932.

Lutensol TDA-9 is a commercially available 9 mole ethoxylate of tridecylalcohol from BASF Corporation, 100 Campus Drive, Florham Park, N.J.07932.

Vantocil P is a commercially available 20% solution of polyhexamethylene biguanide Arch Chemicals, Inc. 5660 New Northside Drive,Suite 1100 Atlanta, Ga. 30328.

A test sample concentrate was prepared according to the invention perbelow:

TS1 50% Bardac 205M 20% Vantocil P 30% Distilled water

TS2 50% Bardac 205M 20% Vantocil P 6% Lutensol TDA-9 8% Trilon M 16%Water

The formulation (TS1) was then tested for bactericidal activity againstPseudomonas aeruginosa using the OECD Quantitative Method for EvaluatingBacteriocidal activity of Microbiocides used on hard, non-poroussurfaces

Microorganism Preparation

Pseudomonas aeruginosa ATCC 15442, was propagated and prepared accordingto the OECD bacteriocidal method. (Centrifuged 10,000 g for 20 minutesand resuspended in 1 mL PBS). Soil Load: 25 μL BSA, 100 μl mucin, 35 μLyeast extract.

Carriers: Magnetized stainless steel disks (1 cm in diameter, 0.7 mmthick)—washed in a 5% DECON-Clean solution, rinsed in deionized water,dried and autoclaved.

Carrier Inoculation: 10 μL of the soil/bacteria mixture was placed inthe center of each carrier. The carriers were dried under vacuum atambient temperature for 1 hour.

Test Substances:

The test substance (TS1) was serially diluted in OECD hard water andlabeled #1-#8.

Sample ID Composition A TS1 @ 8 oz./gal. B TS1 @ 4 oz./gal. C TS1 @ 1.0oz./gal D TS1 @ 0.75 oz./gal E TS1 @ 0.5 oz./gal F TS1 @ 0.25 oz./gal GTS1 @ 1.0 oz./gal plus Lutensol TDA-9 and Trilon M at equal level as inexample 8 H TS2 @ 8.0 oz/gal

-   -   Test Temperature: Ambient (15-30° C.)    -   Exposure Time: 5 minutes    -   Neutralizer: 10 mL of DeyEngly Broth (DE)    -   Media: Tryptic Soy Agar    -   Incubation 35±2° C. for 2 days

General Test Procedure:

Dried, inoculated carriers were placed inside of small plastic medicinejars. 50 μL of diluted test substances was placed on the center of eachdisk at intervals of 15 seconds.

After the specified exposure time (ambient exposure temperature), eachdisk was neutralized at the appropriate interval. Each vial was thenvortexed for 30 seconds. 1.0 mL from the medicine jar (10⁻¹) and 1.0 mLof a 1:100 dilution in Phosphate Buffered Dilution Water (10⁻³) werepour plated, and the remaining was added to a pre-wet (with saline)analytical filter unit. The medicine jar was washed with 20 mL of salinetree times, with each washing added to the filter unit. A magnet wasplaced on the outside of the jar to hold the carrier in the jar whilepouring out the liquid. The liquid was filtered through the membrane viaa vacuum connection, and the funnels were rinsed with 40 mL of salineand then filtered through the membrane. The membranes were asepticallyadded to the appropriate agar medium. Three carriers were used for eachtest condition.

For the carrier enumeration controls, four carriers were used per timepoint. 50 μL of saline was added to each carrier. After the desiredexposure time, 10 mL of the neutralizer was added to each carrier (inthe same medicine jars as above). The carrier enumeration controls werepour plated. The 10⁻², 10⁻³ and 10⁻⁴ dilutions were plated.

Number Centrals Test System C1 C2 C3 C4 Ave P. 553 × 10⁴ 458 × 10⁴ 602 ×10⁴ 550 × 10⁴ 5.4 × 10⁶ aeruginosa

Culture #s Test System CFU/mL P. aeruginosa 351 × 10⁷  3.5 × 10⁹

Pseudomonas Aeruginosa Ave Log % Sample Replicate CFU/CarrierCFU/Carrier Log Reduction Reduction TS1 @ 8 1    72 × 10′    2.8 × 10²2.44 4.29 99.994 oz./gal. 2 30 3 79 TS1 @ 4 1 43    23 × 10² 1.36 5.3799.999 oz./gal. 2 18 3  7 TS1 @ 1.0 1    36 × 10³   3.1 × 10⁴ 4.49 2.2499.42 oz./gal 2    34 × 10³ 3    23 × 10³ TS1 @ 0.75 1    94 × 10³   4.5× 10⁴ 4.65 2.08 99.16 oz./gal 2    244 × 10¹ 3    40 × 10³ TS1 @ 0.5 1   183 × 10³   2.6 × 10⁵ 5.41 1.32 95.18 oz./gal 2    826 × 10³ 3    357× 10³ TS1 @ 0.25 1    567 × 10³ >8.6 × 10⁵ >5.93 <0.80 No reductionoz./gal 2 >1000 × 10³ <84.07% 3 >1000 × 10³ TS1 @ 1.0 1    90 × 10³ <3.0× 10² <2.47 >4.26 >99.994 oz./gal 2  0 plus 3  0 Amine Oxide and EDTA atequal level as in example 8 TS2 @ 8.0 1  0 <1 0 >6.73 >99.99998 oz/gal..2  0 3  0

As can be seen, the addition of surfactant and chelant gave superiorreduction in the presence of Pseudomonas Aeruginosa over thecompositions comprising the quaternary ammonium compound and cationicbiocide alone.

Additional Formulations were Made and Tested as Per Below:

The data outlined in the next 2 tables is outlined in examples #9-#13.Unless we see a reason to break those out as a composite I wouldrecommend that we delete these 2 tables and level the data as disparatewithin the examples. When it comes to examination of the application Ihave specified in the examples the critical comparisons referenced totest ID numbers and example numbers so the support data can be easilyfound.

Trilon M Quat PHMB TDA (Active Versene OECD (ppm) (ppm) (ppm) AO-8 Ppm)100 LR Physical 1 1953 312.5 0 0 3.25 99.94 Clear 2 1953 312.5 500 5002.8 99.84 Clear 3 1953 312.5 500 2000 >6.69 >99.9999 Very cloudy 4 1953312.5 1000 1000 >5.79 >99.999 Slight cloudy 5 1953 312.5 2000 500 2.6599.7 Clear 6 1953 312.5 2000 2000 >6.69 >99.9999 Very cloudy 7 1953312.5 2000 0 2.26 99.46 Clear 8 1953 312.5 0 2000 4.44 99.99 Very cloudy9 1953 312.5 0 0 2.54 99.7 Clear 10 1953 312.5 500 1000 >5.77 >99.999Clear 11 1953 312.5 500 500 3.14 99.9 Clear pH 11 12 1953 312.5 500 5003.6 99.94 Clear EDTA vs trilon 13 1953 312.5 500 2000 >6.07 >99.9999Clear pH 8.5 14 3905 625 500 500 >6.07 >99.9999 Clear 2 oz/gal actives15 1953 312.5 0 500 500 >5.47 >99.9992 Clear Barlox vs. TDA 16 1953312.5 500 500 4.3 99.99 Clear MB50 vs. 205M 0 0 0 0 17 977 312.5 0 0 01.6 97.45 Clear MB50 1.954 1.954 2.579 2.579 18 977 312.5 0 500 500 2.399.5 Clear MB50 0 0 0 0 19 1953 312.5 0 0 0 2.34 99.5 Clear 2 oz/gal0.977 1.954 1.2895 2.579 actives 20 977 312.5 0 500 1000 2.48 99.6 ClearMB50 with 0.977 0.4885 1.2895 0.64475 PG water 21 977 312.5 0 2000 10003.73 99.98 Clear MB50 0.977 3.908 1.2895 5.158 22 977 312.5 0 250 10003.79 99.98 Clear MB50 0.977 1.954 1.2895 2.579 23 977 312.5 0 500 10003.85 99.98 Clear MB50 0.977 0.977 1.2895 1.2895 24 977 312.5 0 1000 10004.81 99.998 Clear MB50

Summary of the Results of the Foregoing Table are Shown Below:

Log Composition MB50 205M PHMB TDA-9 AO-8 Trilon Versene Physical pHreduction Turbid vs. non turbid examples 10 25 10 3.2 16 Clear 10 >5.77 4 25 10 6.4 16 Slight cloudy 10 >5.79 13 25 10 3.2 32 Clear 8.5 >6.07 3 25 10 3.2 32 Very cloudy 10 >6.69 205m vs. MB50 11 25 10 3.2 8 Clear11 3.14 16 25 10 3.2 8 Clear 10 4.3 EDTA vs. Triton  2 25 10 3.2 8 Clear10 2.8 12 25 10 3.2 8 Clear 10 3.6 Ratio chelant to TDA or QAC  2 25 103.2 Clear 10 2.8 12 25 10 3.2 8 Clear 10 3.6 10 25 10 3.2 8 Clear10 >5.77 13 25 10 3.2 16 Clear 8.5 >6.07 32 TDA vs. AO-8 15 25 10 3.2 88 Clear 10 >5.47  2 25 10 3.2 8 Clear 10 2.8As can be seen, the use of amine oxide compared to TDA gave far superiorand more than two logs better reduction in bacteria. Also, thecomparison of ration of chelant to TDA or QAC showed that increasing theratio of chelant to quat by as much as 2 to 3 times greatly improvedreduction on bacteria.

Example 10

The concentration gradient experiment from example 9 demonstrated theability to achieve a high level of microbial efficacy in “wet”applications. However, the levels of chemistry required to achieve therequired levels of efficacy (minimum 3 logs) were at concentrations (TS1@ 4-8 oz/gallon) that were likely to cause problems from a cost and eyeirritancy perspective.

As such we undertook a series of designed experiments to optimize theconcentration of biocidal actives, their preferred embodiment and thecritical concentrations of adjuncts required to achieve a high level ofbactericidal efficacy against gram negative organisms (e.g. Pseudomonasaeruginosa)

Experimentally we composed a series of compositions inclusive ofdifferent levels of chelant and surfactants to determine an optimum.

These solutions were diluted @ a ratio of 1 oz/gallon of water toproduce the active solutions for microbial testing. The compositions inthis experiment when diluted to 1 oz/gal level contained QAC˜1950 ppmand PHMB˜310 ppm.

Raw materials by weight (g) Sample ID QAC PHMB TDA Trilon M Water 112.50 5.00 0.00 0.00 32.50 2 12.50 5.00 1.60 4.00 26.90 3 12.50 5.001.60 16.00 14.90 4 12.50 5.00 3.20 8.00 21.30 5 12.50 5.00 6.40 4.0022.10 6 12.50 5.00 6.40 16.00 10.10 7 12.50 5.00 6.40 0.00 26.10 8 12.505.00 0.00 16.00 16.50

Critical ranges of surfactant and chelant Sample TDA Trilon M 1 0 0 2500 500 3 500 2000 4 1000 1000 5 2000 500 6 2000 2000 7 2000 0 8 0 2000

Concentrate samples 3, 6, 8 separated upon sitting. All samples wereshaken before dilution to ensure uniformity.

Microbiological Test Method: Quantitative Method for EvaluationsBactericidal Activity of Microbiocides Used on Hard, Non-Porous Surfaces(OECD) Rev. 3/10/2010.

-   -   Test Systems: Pseudomonas aeruginosa ATCC 15442    -   Propagated and prepared according to the OECD bacteriocidal        method. (Centrifuged 1000 g for 20 minutes and resuspended in 1        mL PBS).    -   Soil Load: 25 μL BSA, 100 μL mucin, 35 μL yeast extract.    -   Carriers: Magnetized stainless steel disks (1 cm in diameter,        0.7 mm thick)—washed in a 5% DECON-Clean solution, rinsed in        deionized water, dried and autoclaved.    -   Carrier Inoculation: 10 μL of the soil/bacteria mixture was        placed in the center of each carrier. The carriers were dried        under vacuum at ambient temperature for 1 hour.

Test Substances: Test Temperature: Ambient (15-30° C.)

Exposure Time: 5 minutes

Neutralizer: 10 mL of DeyEngly Broth (DE) Media: Tryptic soy Agar

Incubation: 35±2° C. for 2 days

General Test Procedure:

Dried, inoculated carriers were placed inside of small plastic medicinejars. 50 μL of diluted test substance was placed onto the center of eachdisk at intervals of 15 seconds. After the specified exposure time(ambient exposure temperature), each disk was neutralized at theappropriate interval. Each vial was then vortexed for 30 seconds.

1.0 mL from the medicine jar (10⁻¹) and 1.0 mL of a 1:100 dilution inPhosphate buffered Dilution Water (10⁻³) were pour plated, and theremaining was added to a pre-wet (with saline) analytical filter unit.The medicine jar was washed with 20 mL of saline three times, with eachwashing added to the filter unit. A magnet was placed on the outside ofthe jar to hold the carrier in the jar while pouring out the liquid. Theliquid was filtered through the membrane via a vacuum connection, andthe funnels were rinsed with 40 mL of saline and then filtered throughthe membrane. The membranes were aseptically added to the appropriateagar medium. Three carriers were used for each test condition.

For the carrier enumeration controls, four carriers were used per timepoint. 50 μL of saline was added to each carrier. After the desiredexposure time, 10 mL of the neutralizer was added to each carrier (inthe same medicine jars as above). The carrier enumeration controls werepour plated. The 10⁻², 10⁻³ and 10⁻⁴ dilutions were plated.

Culture #s Test System CFU/mL Ave CFU/mL P. aeruginosa 237, 235 × 10⁷2.36 × 10⁹ Number Controls Test System C1 C2 C3 C4 Ave CFU/mL P.aeruginosa 500 × 10⁴ 357 × 10⁴ 606 × 10⁴ 521 × 10⁴ 5.0 × 10⁶ Log 6.69Ave CFU/ Log % Sample Replicate CFU/Carrier Carrier Log ReductionReduction 1 1  70 × 10¹ 2.8 × 10³ 3.44 3.25 99.9 2 232 × 10¹ 3 524 × 10¹2 1 232 × 10¹ 7.8 × 10³ 3.89 2.80 99.8 2 24 (0) 3  21 × 10³ 3 1  0 (0)<1 0 >6.69 >99.9999 2  0 (0) 3  0 (0) 4 1  0 (0) <8 <0.90 >5.79 >99.9992  0 (0) 3 22 (0) 5 1  34 × 10³ 1.1 × 10⁴ 4.04 2.65 99.7 2  7 (0) 3 15(0) 6 1  0 (0) <1 0 >6.69 >99.9999 2  0 (0) 3  0 (0) 7 1  43 × 10³ 2.7 ×10⁴ 4.43 2.26 99.4 2  33 × 10³ 3  7 × 10³ 334 × 10¹ 8 1  55 × 10¹ 1.8 ×10² 2.25 4.44 99.99 2  0 (0) 3  0 (0)

This 1^(st) DOE highlights the importance of inclusion of chelant in thecomposition for enhancement of wet efficacy of the blend of PHMB and QAC(see #1 vs. #8)

It also highlights the detrimental effect of nonionic surfactant in thiscomposition in the absence of chelant (see #1 vs. #7) and importance ofchelant concentration relative to PHMB and QAC (see #2,3,4 and 5)

Example 11

This example entails an expansion of the OCED design outlined in example#10. It included focus on pH of the concentrates, impact of phasestability of the concentrates, choice of chelant for optimum efficacy,selection of benzyle quat vs. a linear quat (Lonza MB50 vs. Lonza 205M)and evaluation of an amine oxide as an alternative to the linearnon-ionic surfactant TDA-9.

Raw materials by weight (g) Bardac Barlox Barquat Sample 205M PHMB TDATrilon M Water AO-8 MB50 EDTA 9 12.50 5.00 0.00 0.00 32.50 10 12.50 5.001.60 8.00 22.90 11 12.50 5.00 1.60 4.00 26.90 pH 11.0 12 12.50 5.00 1.6026.90 4.00 13 12.50 5.00 1.60 16.00 14.90 14 25.00 10.00 1.60 4.00 9.4015 12.50 5.00 4.00 24.50 4.00 16 5.00 1.60 4.00 26.90 12.50 Sample 11adjusted to pH 11 with NaOH. Sample 13 adjusted to pH 8.5 with PhosAcid.

Each of these compositions was diluted 1 oz/gallon with OECD hard waterand evaluated for microbial efficacy against Pseudomonas aeruginosausing the Quantitative Method for evaluations bactericidal activity ofmicrobiocides used on hard, non-porous surfaces (OECD) as outlinedpreviously.

Culture #s Test System CFU/mL P. aeruginosa 34 × 10⁷ Number ControlsTest System C1 C2 C3 C4 Ave CFU/mL P. aeruginosa 126 × 10⁴ 117 × 10⁴ 154× 10⁴ 88 × 10⁴ 1.2 × 10⁶ Log 6.07 Ave Log % Sample Replicate CFU/CarrierCFU/Carrier Log Reduction Reduction  9 1 292 × 10¹   3.4 × 10³ 3.53 2.5499.7 2 226 × 10¹   3  5 × 10³   10 1 0 <2 <0.30 >5.77 >99.999 2 0 3 3 111 242 × 10¹   8.6 × 10² 2.93 3.14 99.9 2 13 3  12 × 10⁻¹ 164(0) 12 1  74× 10¹   3.0 × 10² 2.47 3.60 99.9 2 4 3  19 × 10¹   100(0) 13 1 1 <10 >6.07 >99.9999 2 0 3 0 14 1 0 <1 0 >6.07 >99.9999 2 0 3 0 15 1 2 <4<0.60 >5.47 >99.999 2 8 3 0 16 1  9 × 10¹   6.0 × 10¹ 1.77 4.30 99.99 53(0) 2 80 3 28

The results from these experiments highlight a range of criticaldiscoveries. The clear advantage of amine oxide vs. an alcoholethoxylate surfactant for enhanced efficacy (see #11 vs #15). Theability to markedly reduce the level of QAC and PHMB required to achievea high level of efficacy with optimized concentrations of chelant andsurfactant selection (See #14 vs. #15) and to confirm the critical ratioof chelant to biocide (see #9, 10 and 11)

Example 12

This third set of designed experiments focused on optimization of theratios of critical ingredients identified in Examples #10 and #11. Ithas a focus on the critical level of amine oxide as well as the chelant.

Ideal (g) FMB- Sample QAC PHMB AO8 EDTA PG Ester Water 17 6.25 5.00 0.000.00 0.00 38.75 18 6.25 5.00 2.00 8.00 0.00 28.75 19 6.25 5.00 4.00 8.000.00 26.75 20 6.25 5.00 8.00 8.00 0.00 22.75 21 6.25 5.00 16.00 8.000.00 14.75 22 12.50 5.00 0.00 0.00 0.00 32.50 23 6.25 5.00 4.00 8.003.20 23.55 24 6.25 5.00 4.00 4.00 0.00 30.75

The PG ester was polyaldo 10-L-1 Decaglyceryl Monostearate manufacturesby Lonza Corp.

Each of these compositions was diluted 1 oz/gallon with OECD hard waterand evaluated for microbial efficacy against Pseudomonas aeruginosausing the Quantitative Method for evaluations bactericidal activity ofmicrobiocides used on hard, non-porous surfaces (OECD) as outlinedpreviously.

ppm in final solution following dilution QAC (MB50) PHMB FMB- AO8 EDTAPG Ester 17 1000 312 0 0 0 18 1000 312 250 1000 0 19 1000 312 500 1000 020 1000 312 1000 1000 0 21 1000 312 2000 1000 0 22 1953 312 0 0 0 231000 312 500 1000 500 24 1000 312 500 500 0

Culture #s Test System CFU/mL Ave CFU/mL P. aeruginosa 265, 304 × 10⁷2.8 × 10⁹ Number Controls Test System C1 C2 C3 C4 Ave CFU/mL P.aeruginosa 156 × 10⁹ 186 × 10⁹ 244 × 10⁴ 277 × 10⁴ 2.2 × 10⁶ Log 6.34Ave Log % Sample Replicate CFU/Carrier CFU/Carrier Log ReductionReduction 17 1  64 × 10³ 5.6 × 10⁴ 4.74 1.60 97.45 2 405 × 10¹ 3 146 ×10¹ 18 1  89 × 10¹ 3.6 × 10² 2.55 3.79 99.98 2  19 × 10¹ 3  0(0) 19 1 69 × 10¹ 3.1 × 10² 2.49 3.85 99.98 2  23 × 10¹ 3  0(0) 20 1 76(0) 3.4 ×10¹ 1.53 4.81 99.998 2 25(0) 3  1(0) 21 1 42(0) 4.1 × 10² 2.61 3.7399.98 2  36 × 10¹ 3  82 × 10¹ 22 1  14 × 10³ 1.0 × 10⁴ 4.00 2.34 99.5 2 10 × 10³ 3  6 × 10³ 23 1  5 × 10³ 7.3 × 10³ 3.86 2.48 99.6 2  7 × 10³ 3 10 × 10³ 24 1  9 × 10³ 1.1 × 10⁴ 4.04 2.30 99.5 2  8 × 10³ 3  16 × 10³The results from experiments 17-24 highlight synergy between amine oxideand EDTA in conjunction with PHMB and QAC (see #17 vs. #22, #23 and #24)

Example 13 Evaluates the Impact of Surfactant and Chelant on thePersistent Antimicrobial Effects

The test method is based on the EPA method for testing dried chemicalresidues on hard, non-porous surfaces (modified).

-   Test Substances: TS3 @ 0.25 oz/gal, 0.5 oz/gal and 1.0 oz/gal.    -   TS4 @ 0.25 oz/gal, 0.5 oz/gal and 1.0 oz/gal.

TS3 50% Bardac 205M 20% Vantocil P 30% Distilled water

TS4 50% Bardac 205M 20% Vantocil P 6% Lutensol TDA-9 8% Trilon M 16%Water

-   Test Substance Diluent: 400 ppm hard water-   Dilutions: 1.0 oz/gal.    -   1/128=0.00781×100 mL=0.781.    -   0.00781×150 mL=1.17 g/150 mL.    -   0.5 oz/gal solution was made by diluting 50 g of 1 oz/gal with        50 g of hard water.    -   0.25 oz/gal solution was made by diluting 50 g of 1 oz/gal with        50 g hard water.-   Test System: (18-24 hr. AOAC NB) Pseudomonas aeruginosa ATCC 15442.    -   Prep—decant culture—not disturbing pellicle, vortex and then        centrifuge at 10,000 g for 15 minutes and re-suspended in 10 mL        PBS.-   Organic Soil Load: 5% fetal bovine serum-   Carriers: 1″×1″ glass microscope slides-   Test Substance Application: 50 mL of the test substance was spot    inoculated over the carrier in as many spots as possible. The    carriers were allowed to dry for 24 hours in disposable Petri dishes    in the bio-safety hood/turned off with the lids cracked.-   Wears: The test surfaces were subjected to 30 abrasive wears using a    dry cloth on the gardner abrasion tested per the EPA methodology.    This was immediately followed by Application of a 10 uL bacterial    inoculum to the carrier surface. After 10 minutes the carrier was    neutralized and any survivors were counted.-   Neutralizers: 20 mL DE broth-   Dilutions Plated: Inoculation 10⁶ and 10⁷ in duplicate.    -   Test Carriers: Direct, 10² and 10⁴ in duplicate.    -   Control Carriers: Direct, 10², 10³, and 10⁴ in duplicate.-   Plating Medium: Tryptone Glucose Extract Agar-   Incubation: 35+/−° C. for 48 hours

Culture Nos. Test System CFU/mL Ave CFU/mL P. aeruginosa 118 and 122 ×10⁷ 1.2 × 10⁹

Control Carriers Numbers CFU/Carrier Test System Wears Replicate CFU/mL(×20) Log Ave P. aeruginosa 30 1 164, 136 × 10³ 3.0 × 10⁶ 6.47 6.46 2162, 127 × 10³ 2.9 × 10⁶ 6.46

Test Carriers-TS3 Test CFU/Carrier Log Log Substance Wear ReplicateConc. CFU/mL (×20) Ave Reduction P. aeruginosa 30 1 0.25 oz/gal 5, 5 (0)1.0 × 10² 2.21 4.25 2 13, 14 (0) 2.7 × 10² P. aeruginosa 30 1  0.5oz/gal 1, 0 (0) <20 <1.57 >4.89 2 4, 3 (0) 7.0 × 10¹ P. aeruginosa 30 1 1.0 oz/gal 0, 0 (0) <20 <1.30 >5.16 2 1, 0 (0) <20

Test Carriers-TS4 Test CFU/Carrier Log Log Substance Wear ReplicateConc. CFU/mL (×20) Ave Reduction P. aeruginosa 30 1 0.25 oz/gal 324, 303(0) 6.1 × 10³ 4.29 2.17  4, 2 × 10² 2 25, 39 × 10² 6.4 × 10⁴ P.aeruginosa 30 1  0.5 oz/gal 142, 146 (0) 2.9 × 10³ 4.00 2.46 2 18, 18 ×10² 3.6 × 10⁴ P. aeruginosa 30 1  1.0 oz/gal 9, 10 (0) 1.9 × 10² 3.373.09 2 15, 15 × 10² 3.0 × 10⁴

The comparison of TS3 to TS4 provides 2 key pieces of information. Itfirst highlights the lower limits of PHMB and QAC where we are stillable to achieve at least a 4 log reduction following abrasive wear (e.g.TS3 diluted 0.25 oz/gallon˜975 ppm QAC and ˜156 ppm PHMB)

However when testing the TS4 composition (inclusive of the lutensolTDA-9 surfactant and Trilon M builder) the residual efficacy is markedlyreduced relative to the actives alone. This highlights the negativeimpact that surfactant and chelant choice and levels can have onresidual efficacy.

Example 14

Once critical levels of active and negative effects of builder weredetermined we took the next step of evaluating the system with the best“wet” for residual efficacy relative to the example containing TDA andTrilon as outlined in examples 10 and 13.

Our focus was on the mixture of QAC, PHMB, AO-8 and EDTA.

Formulations EDTA Conc AO-8 conc MB50 PHMB (ppm) (ppm) 12837-42.1 3900625 0 0 12837-42.2 3900 625 0 500 12837-42.3 3900 625 0 1000 12837-42.43900 625 500 0 12837-42.5 3900 625 500 500 12837-42.6 3900 625 500 100012837-42.7 3900 625 1000 0 12837-42.8 3900 625 1000 500 12837-42.9 3900625 1000 1000

These solutions were testing using the same procedure as outlined inExample #13.

Culture Nos. Test System CFU/mL P. aeruginosa 90, 82 × 10⁷ 5:1 152, 160× 10⁶

Control Carriers CFU/Carrier Test System Wear Replicate CFU/mL (×20) LogAve P. aeruginosa 30 1 119, 173 × 10³ 2.9 × 10⁶ 6.46 6.45 2 123, 156 ×10³ 2.8 × 10  6.44 Test CFU/Carrier Log Substance Wear Replicate CFU/mL(×20) Log Ave Reduction 12837-42.1 30 1 1, 1 (0) 20 1.30 1.45 5.00 2 2,0 (0) 40 1.60 12837-42.2 30 1 0, 0 (0) <20 <1.30 <1.30 >5.15 2 0, 0 (0)<20 <1.30 12837-42.3 30 1 0, 0 (0) <20 <1.30 <1.65 >4.80 2 5, 5 (0)   1× 10² 2.00 12837-42.4 30 1 1, 1 (0) 20 1.30 <1.30 5.15 2 0, 0 (0) <20<1.30 12837-42.5 30 1 14, 11 (0) 2.4 × 10² 2.38 2.59 3.86 2 30, 35 (0)6.5 × 10² 2.81 12837-42.6 30 1 167, 175 (0) 3.4 × 10³ 3.53 2.56 3.89 22, 1 (0) 40 1.60 12837-42.7 30 1 0, 0 (0) <20 <1.30 <1.45 >5.00 2 2, 0(0) 40 1.60 12837-42.8 30 1 6, 3 (0) 80 1.90 2.10 4.35 2 15, 5 (0)   2 ×10² 2.30 12837-42.9 30 1 6, 6 (0) 1.2 × 10² 2.07 2.77 3.68 2 156, 147(0) 3.0 × 10³ 3.47

Result summary QAC/PHMB EDTA Conc AO-8 conc Log (ppm) (ppm) (ppm)reduction 1 3900/625 0 0 5.00 2 3900/625 0 500 >5.15 3 3900/625 01000 >4.80 4 3900/625 500 0 >5.15 5 3900/625 500 500 3.86 6 3900/625 5001000 3.89 7 3900/625 1000 0 >5.00 8 3900/625 1000 500 4.35 9 3900/6251000 1000 3.68

These results were markedly superior to the composition from example #13based on Lutensol and Trilon M. But surprisingly, the experimentindicated that the combination of EDTA with AO-8 provided less residualefficacy that compositions containing those materials individually (See#2,3,4 and #7).

Example 15

To understand the results from experiment #14 we ran an additionalexperiment using this same methodology with one critical difference. Inthis experiment we compared the efficacy of a treated carrier inoculatedwith 10 uL of bacterial inoculum vs. one treated with 10 uL of inoculumwith 40 uL of added water making the total inoculum volume 50 uL. Thenet effect of this was a 5× dilution of the chemistry on the surface ofthe treated carrier.

This test is driven by the standard methodology. The normal test methodcalls for addition of 50 uL of chemistry to our 1″×1″ carrier. Thatchemistry is dried and subsequently abraded to remove a portion of thechemistry from that surface (simulating wear that would occur if atreated surface were touched many times). This worn surface is theninoculated with 10 uL of a bacterial suspension where it reacts on thatsurface for 10 minutes. The critical factor here is the addition of 10uL of inoculum to the dried residue of 50 uL of chemistry.

Because of this when the inoculum re-hydrates the chemical residue itrepresents as much as a 5× increase in the concentration of actives. Thesolutions in example #13 and #14 indicate that both AO-8, EDTA andLutensol TDA-9 have a detrimental effect on the residual efficacy ofPHMB/QAC during this concentration effect. Though AO-8 is less affectedthan TDA9 making selection of that ingredient more appropriate for amixture that provides both good “wet” and “dry’ residual activity.

That said an experiment where we re-hydrate our chemistry with 50 uL vs.10 uL helps us both understand the mechanism and defines lower limits ofchemistry where acceptable efficacy can be found. The test compositionwe chose for this experiment is outlined below.

Test composition:

EDTA Conc AO-8 conc MB50 PHMB (ppm) (ppm) 13137.9.1 3900 625 1000 1000This solution was dried onto a carrier and challenged with 10 uL or 50uL of bacterial inoculum with the results outlined below

Test CFU/Carrier Log Substance Wear Replicate CFU/mL (×20) Log AveReduction 13137-9.1 30 1 66, 63 (0) 1.3 × 10³ 3.11 2.62 3.86 10 ul 2 8,6 (0) 1.4 × 10² 2.14 inoculum 13137-9.1 30 1 0, 0 (0) <20 <1.30<1.30 >5.15 50 uL 2 0, 0 (0) <20 <1.30 inoculum

Example 16

Because disinfectants are used in a variety of different areas it iscritical to develop compositions that are minimally irritating to theworkers who use them as part of their daily routine. One area ofparticular concern is the potential eye irritancy of a disinfectantcomposition. This becomes particularly important when we look at aproduct that is intended to provide a semi-durable residue ondisinfected surfaces as that may require higher levels of activeingredients than would be required for a conventional disinfectant.

As part of our development process we discovered that we could achievevery effect “wet” and “dry” residual efficacy with a range ofcompositions. But as the development progressed it became clear thatthere was a range of ingredients and concentration ranges that werepreferred due to their low irritancy.

Compositions:

Raw Material 12837.39.1 12837.39.2. 12837.39.3. 12837.36.1 Water (DT)  6g 23 g 23 g 23 g Luntensol  6 g TDA-9 Lonza MB-50 50 g 25 g — — Lonza205M — — 25 g 50 g Vantocil P 20 g 20 g 20 g 20 g Lonza AO-8  8 g 16 g16 g — Versene 100 16 g 16 g 16 g — Trilon M — — — 16 g

Each of these compositions was diluted at a rate of 2 oz/gal before theywere tested using the Epioccular model.

Methodology:

The MatTek EpiOcular™ model can be used to assess the potential ocularirritation of test articles by determining the cell viability of thetissue after exposure to the test articles. The objective of this studywas to assess the ocular irritation potential of 1.56% (w/w) 12837.36.1.Control and test article exposure times were 5 minutes, 10 minutes, 20minutes, 40 minutes and 60 minutes. After the exposures MTT wasperformed and data was normalized to the negative control, sterileultrapure water (water purified and deionized ˜18.2 MΩ-cm). The positivecontrol, 10% benzalkonium chloride (BC), was effective, reducing cellviability to <10% of control for all exposure times (Tables 2a-e) andFIG. 1). The relative cell viability of ocular tissues treated with1.56% (w/w) 12837.36.1 was 29.7%, 26.8%, 15.3%, 8.9% and 8.0% after 5,10, 20, 40 and 60 minute exposures, respectively (Tables 2a-e and FIG.1), resulting in an ET₅₀ of 1.54 minutes and an estimated in vivo Draizescore of 77.2 (Table 1). Because of the results of this study, 1.56%(w/w) 12837.36.1 was categorized as a severe/extreme ocular irritant.

TABLE 1 Ocular Irritancy categorization. ET₅₀ Estimated (min) DraizeCategorization 10% Benzalkonium Cl 0.03 >110 Severe/Extreme Irritant1.56% (w/w) 12837.36.1 1.54 77.2 Severe/Extreme Irritant

The MatTek EipOcular™ model can be used to assess the potential ocularirritation of test articles by determining the cell viability of thetissue after exposure to the test articles. The objective of this studywas to assess the ocular irritation potential of 1.56% (w/w) 12837.39.1.Control and test article exposure times were 5 minutes, 10 minutes, 20minutes, 40 minutes and 60 minutes. After the exposures MTT wasperformed and data was normalized to the negative control, sterileultrapure water (water purified and deionized ˜18.2 MΩ-cm). The positivecontrol, 10% benzalkonium chloride (BC), was effective, reducing cellviability to <10% of control for all exposure times (Tables 2a-e) andFIG. 1). The relative cell viability of ocular tissues treated with1.56% (w/w) 12837.39.1 was 87.5%, 48.8%, 32.9%, 17.3% and 12.8% after 5,10, 20, 40 and 60 minute exposures, respectively (Tables 2a-e and FIG.1), resulting in an ET₅₀ of 9.24 minutes and an estimated in vivo Draizescore of 28.7 (Table 1). Because of the results of this study, 1.56%(w/w) 12837.39.1 was categorized as a moderate ocular irritant.

TABLE 1 Ocular Irritancy categorization ET₅₀ Estimated (min) DraizeCategorization 10% Benzalkonium Cl 0.03 >110 Severe/Extreme Irritant1.56% (w/w) 12837.39.1 9.24 28.7 Moderate Irritant

The MatTek EipOcular™ model can be used to assess the potential ocularirritation of test articles by determining the cell viability of thetissue after exposure to the test articles. The objective of this studywas to assess the ocular irritation potential of 1.56% (w/w) 12837.39.2.Control and test article exposure times were 5 minutes, 10 minutes, 20minutes, 40 minutes and 60 minutes. After the exposures MTT wasperformed and data was normalized to the negative control, sterileultrapure water (water purified and deionized ˜18.2 MΩ-cm). The positivecontrol, 10% benzalkonium chloride (BC), was effective, reducing cellviability to <10% of control for all exposure times (Tables 2a-e) andFIG. 1). The relative cell viability of ocular tissues treated with1.56% (w/w) 12837.39.2 was 103.5%, 90.2%, 75.1%, 45.0% and 19.7% after5, 10, 20, 40 and 60 minute exposures, respectively (Tables 2a-e andFIG. 1), resulting in an ET₅₀ of 36.43 minutes and an estimated in vivoDraize score of 12.1 (Table 1). Because of the results of this study,1.56% (w/w) 12837.39.2 was categorized as a minimal to non-ocularirritant.

TABLE 1 Ocular Irritancy categorization ET₅₀ Estimated (min) DraizeCategorization 10% Benzalkonium Cl 0.03 >110 Severe/Extreme Irritant1.56% (w/w) 12837.39.2 36.43 12.1 Minimal to Non-Irritant

The MatTek EpiOcular™ model can be used to assess the potential ocularirritation of test articles by determining the cell viability of thetissue after exposure to the test articles. The objective of this studywas to assess the ocular irritation potential of 1.56% (w/w) 12837.39.3.Control and test article exposure times were 5 minutes, 10 minutes, 20minutes, 40 minutes and 60 minutes. After the exposures MTT wasperformed and data was normalized to the negative control, sterileultrapure water (water purified and deionized ˜18.2 MΩ-cm). The positivecontrol, 10% benzalkonium chloride (BC), was effective, reducing cellviability to <10% of control for all exposure times (Tables 2a-e) andFIG. 1). The relative cell viability of ocular tissues treated with1.56% (w/w) 12837.39.3 was 69.6%, 73.5%, 62.5%, 25.3% and 17.2% after 5,10, 20, 40 and 60 minute exposures, respectively (Tables 2a-e and FIG.1), resulting in an ET₅₀ of 23.36 minutes and an estimated in vivoDraize score of 12.1 (Table 1). Because of the results of this study,1.56% (w/w) 12837.39.3 was categorized as a mild ocular irritant.

TABLE 1 Ocular Irritancy categorization ET₅₀ Estimated (min) DraizeCategorization 10% Benzalkonium Cl 0.03 >110 Severe/Extreme Irritant1.56% (w/w) 12837.39.3 23.36 16.3 Mild Irritant

Conclusion:

To be defined as a sanitizer, the test substances on the hard inanimatesurface must reduce the total number of organisms by at least 99.9%(based on the Geometric Mean) on the surface within a 5 minute period(after the final inoculation).

The antimicrobial efficacy of the commercial disinfectant dropped offwith continual wearing of the treated surface. After 10 and 20 wears, agreater than 99.9% reduction in S. aureus numbers were observed, butfell below acceptable levels over 20 wears.

The antimicrobial efficacy of the QAC blend alone showed variableresults. QAC showed some efficacy with all wear cycles, but a greaterthan 99.9% reduction in S. aureus numbers was only observed with the 30wear testing.

The antimicrobial efficacy of the PHMB alone showed little to noefficacy again S. aureus with any of the wear cycles tested.

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims. Thus, manymodifications and other embodiments of the invention will come to mindto one skilled in the art to which this invention pertains having thebenefit of the teachings presented in the foregoing descriptions and theassociated drawings. Therefore, it is to be understood that theinvention is not to be limited to the specific embodiments disclosed andthat modifications and other embodiments are intended to be includedwithin the scope of the appended claims.

What is claimed is:
 1. An anti-microbial composition comprising: aquaternary ammonium compound; and a cationic biocide, wherein the amountof cationic biocide is less than 1 to 10 in a ratio of cationic biocideto quaternary ammonium on an actives weight basis.
 2. The anti-microbialcomposition of claim 1 wherein the ratio of cationic biocide toquaternary ammonium is from about 1:2.5 to about 1:6.25
 3. Theanti-microbial composition of claim 1 wherein said ratio of cationicbiocide to quaternary ammonium is from about 1:1 to 1:6.25.
 4. Theanti-microbial composition of claim 1 wherein said ratio of cationicbiocide to quaternary ammonium is from about 1:0.625 to 1:10.
 5. Theanti-microbial composition of claim 1 wherein said quaternary ammoniumcompound is in an amount of about 975 to about 6250 ppm.
 6. The canti-microbial composition of claim 1 wherein said cationic biocide isin an amount of about 156 to about 2500 ppm.
 7. The anti-microbialcomposition of claim 1 wherein said cationic biocide is polyhexmethylenebiguanide (PHMB).
 8. The anti-microbial composition of claim 1 whereinsaid quaternary ammonium compound is quaternary ammonium chloride. 9.The anti-microbial composition of claim 1 wherein said compositioncomprises more than one quaternary ammonium compound.
 10. Ananti-microbial composition comprising: a quaternary ammonium compound: acationic biocide, an amine oxide surfactant, and a chelant.
 11. Theanti-microbial composition of claim 10 wherein the ratio of quaternaryammonium to surfactant is from about 2:1 to about 7:1 respectively. 12.The anti-microbial composition of claim 10 wherein the ratio ofquaternary ammonium compound to chelant is from about 2:1 to about 3.5to 1 respectively.
 13. The anti-microbial composition of claim 10wherein said amine oxide has a carbon chain length of 8 or less.
 14. Theanti-microbial composition of claim 10 wherein said chelant is EDTA. 15.The anti-microbial composition of claim 10 wherein said compositioncomprises from about 1.25% to about 50% wt. % of quaternary ammoniumcompound, from about 0.125% to about 8.0 wt. % of cationic biocide, fromabout 0.4% to about 12.8 wt. % of amine oxide, and from about 0.4% toabout 12.8% chelant.
 16. The composition of claim 15 wherein saidconcentrate is diluted to a use composition at a ratio of about 1:64.17. The use solution of claim 16 wherein said solution comprises fromabout 195 ppm to about 7800 ppm of quaternary ammonium compound, fromabout 19.5 ppm to about 1250 ppm of cationic biocide, from about 62.5 toabout 2000 ppm amine oxide, and from about 62.5 to about 2000 ppmchelant.
 18. The anti-microbial composition of claim 10 wherein saidcationic biocide is polyhexmethylene biguanide (PHMB).
 19. Theanti-microbial composition of claim 1 wherein said quaternary ammoniumcompound is quaternary ammonium chloride.
 20. The anti-microbialcomposition of claim 1 wherein said composition provides mild to non-eyeirritancy.
 21. The antimicrobial composition of claim 1 wherein saidcomposition provides wet and dry efficacy.
 22. The antimicrobialcomposition of claim 1 wherein said composition is optimized for non-eyeirritancy, with wet and dry efficacy and reduction in microbialcontamination.
 23. The composition of claim 1 wherein said compositionprovides 99.9% log reduction in microbial contamination following a 24wear cycles abrasion challenge.
 24. A method of disinfecting a surfacecomprising: Applying the disinfectant solution to a surface cleaning,providing wet disinfection and applying a protective coating to the saidsurface a composition according to claim 10, so that a film is formed onsaid surface that continues to reduce microbial contamination aftergeneral wear for an extended period of time.
 25. A method according toclaim 20 wherein the film provides a 99% reduction of Pseudomonasaeruginosa and Staphylococcus aureus following 30 abrasive wear cycles.26. The method of claim 24 wherein said antimicrobial compositionprovides mild to non-eye irritancy.
 27. The method of claim 24 whereinsaid antimicrobial composition of claim 24 wherein said compositionprovides wet and dry efficacy.
 28. The method of claim 24 wherein saidcomposition is optimized for non-eye irritancy, with wet and dryefficacy and reduction in microbial contamination.
 29. The method ofclaim 24 wherein said composition provides 99.9% log reduction inmicrobial contamination following a 24 wear cycle abrasion challenge.30. A method of making an anti-microbial composition that isnon-eye-irritating, and provides both wet and dry efficacy comprising:mixing a solution comprising: a quaternary ammonium compound; a cationicbiocide, an amine oxide surfactant, and a chelant wherein the ratio ofquaternary ammonium to surfactant is from about 2:1 to about 7:1respectively.
 31. The method of claim 30 wherein said amine oxide has acarbon chain length of 8 or less.